Novel 13-transmembrane protein expressed in prostate cancer

ABSTRACT

A novel prostate tumor associated gene (designated 24P4C12) and its encoded protein is described. 24P4C12 is highly expressed in prostate tissue xenografts, providing evidence that it is turned on in at least some prostate cancers. 24P4C12 provides a diagnostic and/or therapeutic target for prostate and other cancers.

[0001] This application claims the benefit of U.S. provisionalapplication serial No. 60/128,858, filed Apr. 12, 1999, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention described herein relates to a novel gene and itsencoded protein, termed 24P4C12, and to diagnostic and therapeuticmethods and compositions useful in the management of various cancersthat express 24P4C12, particularly prostate cancers.

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of human death next tocoronary disease. Worldwide, millions of people die from cancer everyyear. In the United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

[0004] Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

[0005] Worldwide, prostate cancer is the fourth most prevalent cancer inmen. In North America and Northern Europe, it is by far the most commonmale cancer and is the second leading cause of cancer death in men. Inthe United States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

[0006] On the diagnostic front, the lack of a prostate tumor marker thatcan accurately detect early-stage, localized tumors remains asignificant limitation in the management of this disease. Although theserum PSA assay has been a very useful tool, its specificity and generalutility is widely regarded as lacking in several important respects.

[0007] Progress in identifying additional specific markers for prostatecancer has been improved by the generation of prostate cancer xenograftsthat can recapitulate different stages of the disease in mice. The LAPC(Los Angeles Prostate Cancer) xenografts are prostate cancer xenograftsthat have survived passage in severe combined immune deficient (SCID)mice and have exhibited the capacity to mimic disease progression,including the transition from androgen dependence to androgenindependence and the development of metastatic lesions (Klein et al.,1997, Nat. Med.3:402). More recently identified prostate cancer markersinclude PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735), and STEAP (Hubert et al., 1999, Proc. Natl.Acad. Sci. USA 96: 14523).

[0008] While previously identified markers such as PSA, PSM, PCTA andPSCA have facilitated efforts to diagnose and treat prostate cancer,there is need for the identification of additional markers andtherapeutic targets for prostate and related cancers in order to furtherimprove diagnosis and therapy.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a novel family of genes andproteins, characterized by multiple transmembrane regions and expressionin prostate cancer. More particularly, the invention provides a novelgene and protein, termed 24P4C12. The 24P4C12 gene encodes a 710 aminoacid protein containing 13 transmembrane domains and bearing homology tomurine and C. elegans genes containing 12 transmembrane domains. Thenucleotide and encoded amino acid sequences of the entire coding andpartial non-coding regions of the human 24P4C12 gene are shown in FIGS.1A-1D (SEQ ID NOS: 1, 2). RT-PCR and Northern blot analyses showexpression of 24P4C12 in normal colon, prostate, kidney and lung, and inprostate cancer xenografts. The transmembrane nature of the 24P4C12protein, combined with its expression in prostate cancer, suggest that24P4C12 is a target for prostate cancer therapy using, for example,antibodies and other small molecules capable of binding to andmodulating the 24P4C12 protein in vivo. In addition, because of itslocation on the cell surface of prostate cancer cells, antibodies andother agents capable of detecting 24P4C12 protein can be useful inprostate cancer imaging methods. Various other molecular detectionassays using, for example, polynucleotide probes and primers capable ofdetecting 24P4C12 transcription products, may also find use indiagnosing, monitoring, prognosing, and staging prostate cancer andpotentially other cancers.

[0010] The invention provides polynucleotides corresponding orcomplementary to the 24P4C12 gene, mRNA, or fragments thereof, includingcDNAs, RNAs, oligonucleotide probes, and primers. The invention furtherprovides methods for detecting the presence of 24P4C12 polynucleotidesin various biological samples. Molecular diagnostic assays for prostatecells using 24P4C12 polynucleotides are also provided. Such assays mayprovide diagnostic and/or prognostic information concerning the presenceand degree of prostate cancer. The invention further provides means forisolating cDNAs and the gene encoding 24P4C12, as well as those encodingmutated and other forms of 24P4C12. Recombinant DNA molecules containing24P4C12 polynucleotides, cells transformed or transduced with suchmolecules, and host-vector systems for the expression of 24P4C12 geneproducts are also provided. The invention further provides 24P4C12proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 24P4C12 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker.

[0011] The invention further provides methods for detecting the presenceand status of 24P4C12 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 24P4C12.A typical embodiment of this invention provides methods for monitoring24P4C12 gene products in a tissue sample having or suspected of havingsome form of growth disregulation such as cancer.

[0012] The invention further provides various therapeutic compositionsand strategies for treating cancers that express 24P4C12 such as cancerof the prostate, including therapies aimed at inhibiting thetranscription, translation, processing or function of 24P4C12 as well ascancer vaccines.

[0013] In addition, the invention provides a novel gene and proteinrelated to 24P4C12, termed H38087. The H38087 gene encodes a 704 aminoacid protein containing 11 potential transmembrane domains. Thenucleotide and encoded amino acid sequences of the entire coding andpartial non-coding regions of the human H38087 gene are shown in FIGS.7A-7D (SEQ ID NOS: 6, 7). The 58 base pairs of 5′ untranslated regionare very GC rich (87%), indicating that this gene may containtranslational regulatory elements. The amino acid sequences of 24P4C12and H38087 are 44% identical and 56% homologous over the entire sequence(FIG. 8). Expression analysis shows that H38087 is ubiquitouslyexpressed (FIG. 9), with highest expression levels detected in testis.Expression is also observed in each of the various LAPC xenograftsexamined. H38087 could serve as a control for testing 24P4C12-specifictherapeutics, or provide a diagnostic and/or therapeutic target. Atherapeutic that selectively affects 24P4C12, but not H38087, may beless toxic to normal cells. Therefore, H38087 protein may be useful as apre-clinical testing tool for therapeutic modalities directed towards24P4C12. H38087 protein expression, however, may be less ubiquitous thanits RNA expression, suggesting H38087 as a target for diagnostic andtherapeutic strategies.

[0014] The invention additionally provides a method for identifying a24P4C12 specific binding agent. The method comprises contacting acandidate agent that binds 24P4C12 with H38087, and determining whetherthe candidate agent binds H38087. A lack of binding of the candidateagent to H38087 being indicative of 24P4C12 specificity. Such bindingcan be detected using conventional binding assays known in the art,including representative assays described herein.

BRIEF DESCRIPTION OF THE FIGURES

[0015] FIGS. 1A-1D. Nucleotide (SEQ ID NO: 1) and deduced amino acid(SEQ ID NO: 2) sequences of entire coding region (and part of the 3′non-coding region) of the 24P4C12 gene. This sequence was generated fromthe overlapping sequences of three cDNA clones, designated 24P4C12-GTE9,24P4C12-GTE5 and 24P4C12-GTE4 (Example 2). Thirteen potentialtransmembrane domains are underlined in bold. A Kozak sequence andputative start methionine are indicated in bold.

[0016]FIG. 1E. Nucleotide (SEQ ID NO: 3) and ORF amino acid (SEQ ID NO:4) sequences of the initially isolated SSH fragment of the 24P4C12 gene.

[0017]FIG. 2A. RT-PCR analysis of 24P4C12 gene expression in prostatecancer xenografts, normal prostate, and other tissues and cell lines,showing approximately equal levels of expression in normal prostate andthe LAPC prostate cancer xenografts. Lanes represent the followingtissues: (1) brain; (2) prostate; (3) LAPC-4 AD; (4) LAPC-4 AI; (5)LAPC-9 AD; (6) HeLa; (7) murine cDNA; and (8) negative control.

[0018]FIG. 2B. RT-PCR analysis of 24P4C12 gene expression in varioustissues, showing detectable expression only in normal kidney and lungafter 25 cycles of PCR amplification. Lower level expression isdetectable in a variety of other tissues after 30 cycles ofamplification. Lanes represent the following tissues: (1) brain; (2)heart; (3) kidney; (4) liver; (5) lung; (6) pancreas; (7) placenta; and(8) skeletal muscle.

[0019]FIG. 2C. RT-PCR analysis of 24P4C12 gene expression in varioustissues, showing detectable expression only in normal colon and prostateafter 25 cycles of PCR amplification. Lower level expression isdetectable in a variety of other tissues after 30 cycles ofamplification. Lanes represent the following tissues: (1) colon; (2)ovary; (3) leukocytes; (4) prostate; (5) small intestine; (6) spleen;(7) testis; and (8) thymus.

[0020]FIG. 3A. Northern blot analysis of 24P4C12 expression across apanel of, normal human tissues, showing expression of an approximately 3kb transcript in kidney. Lanes represent the following tissues: (1)heart; (2) brain; (3) placenta; (4) lung; (5) liver; (6) skeletalmuscle; (7) kidney; and (8) pancreas.

[0021]FIG. 3B. Northern blot analysis of 24P4C12 expression across apanel of normal human tissues, showing expression of an approximately 3kb transcript in prostate and colon. Lanes represent the followingtissues: (1) spleen; (2) thymus; (3) prostate; (4) testis; (5) ovary;(6) small intestine; (7) colon; and (8) leukocytes.

[0022]FIG. 3C. Northern blot analysis of 24P4C12 expression in prostatecancer xenografts and prostate cancer cell lines. Lanes represent thefollowing tissues: (1) PrEC; (2) LAPC-4 AD; (3) LAPC-4 AI; (4) LAPC-9AD; (5) LAPC-9 AI; (6) LNCaP; (7) PC-3; (8) DU145; (9) TsuPr1; and (10)LAPC-4 CL.

[0023] FIGS. 4A-4B. Amino acid sequence alignment of the 24P4C12 geneproduct and murine NG22 (SEQ ID NO: 5).

[0024]FIG. 5. Expression of 24P4C12 in LAPC xenografts. RNA wasextracted from the LAPC xenograft that were grown subcutaneously (sc) orintra-tibially (it) within the mouse bone. Northern blots with 10 μg oftotal RNA/lane were probed with the 24P4C12 SSH fragment. Size standardsin kilobases (kb) are indicated on the side. Lanes represent thefollowing tissues: (1) LAPC-4 AD sc; (2) LAPC-4 AD sc; (3) LAPC-4 AD sc;(4) LAPC-4 AD it; (5) LAPC-4 AD it; (6) LAPC-4 AD it; (7) LAPC-4 AD²;(8) LAPC-9 AD sc; (9) LAPC-9 AD sc; (10) LAPC-9 AD it; (11) LAPC-9 ADit; (12) LAPC-9 AD it; (13) LAPC-3 AI sc; and (14) LAPC-3 AI sc.

[0025]FIG. 6A. Expression of 24P4C12 in prostate cancer patient samples.RNA was extracted from the prostate tumors and their normal adjacenttissue derived from prostate cancer patients. Northern blots with 10 μgof total RNA/lane were probed with the 24P4C12 SSH fragment. Sizestandards in kilobases (kb) are indicated on the side. Lanes representthe following tissues: (1) Patient 1, normal adjacent tissue; (2)Patient 1, Gleason 7 tumor; (3) Patient 2, normal adjacent tumor; (4)Patient 2, Gleason 9 tumor; (5) Patient 3, normal adjacent tissue; (6)Patient 3, Gleason 7 tumor.

[0026]FIG. 6B. Expression of 24P4C12 in prostate cancer patient samplesas described for FIG. 6A was compared to β-actin. Lanes represent thefollowing tissues: (1) Patient 1, normal adjacent tissue; (2) Patient 1,Gleason 7 tumor; (3) Patient 2, normal adjacent tumor; (4) Patient 2,Gleason 9 tumor; (5) Patient 3, normal adjacent tissue; (6) Patient 3,Gleason 7 tumor.

[0027] FIGS. 7A-7D. The cDNA (SEQ ID NO: 6) and amino acid (SEQ ID NO:7) sequence of H38087 (clone GTB6). A GC rich (87% GC content) region inthe 5′ untranslated (UTR) region is shown prior to the potential Kozaksequence and start methionine, which are indicated in bold. Thepotential transmembrane domains are underlined in bold.

[0028]FIG. 8. Homology alignment of 24P4C12 with H38087 using the BLASTfunction (NCBI).

[0029]FIG. 9A. Expression of 24P4C12 in human tissues. A multiple tissuenorthern blot (Clontech) with 2 μg of mRNA/lane was probed with the24P4C12 SSH fragment. Size standards in kilobases (kb) are indicated onthe side. Lanes represent the following tissues: (1) heart; (2) brain;(3) placenta; (4) lung; (5) liver; (6) skeletal muscle; (7) kidney; and(8) pancreas.

[0030]FIG. 9B. Expression of 24P4C12 in human tissues. A multiple tissuenorthern blot (Clontech) with 2 μg of mRNA/lane was probed with the24P4C12 SSH fragment. Size standards in kilobases (kb) are indicated onthe side. Lanes represent the following tissues: (1) spleen; (2) thymus;(3) prostate; (4) testis; (5) ovary; (6) small intestine; (7) colon; and(8) leukocytes.

[0031]FIG. 9C. Expression of 24P4C12 in human tissues. An LAPC xenograftnorthern blot with 10 μg of total RNA/lane was probed with the 24P4C12SSH fragment. Size standards in kilobases (kb) are indicated on theside. Lanes represent the following tissues: (1) PC-3; (2) LAPC-4 AD;(3) LAPC4 AI; (4) LAPC-9 AD; (5) LAPC-9 AI.

[0032]FIG. 10A. Detection of 24P4C12 protein in 293T cells transfectedwith 24P4C12 cDNA by 24P4C12-specific polyclonal antibodies. 293T cellswere transiently transfected with empty vector, or 24P4C12 cDNA in pCDNA3.1 CMV-driven MYC-His or pSR-alpha retroviral expression vectors. Celllysates in sample buffer were then subjected to mild heat denaturation(70° C.) and separated on a 10% SDS-PAGE gel and transferred tonitrocellulose. Membranes were then subjected to western analysis with 2μg/ml of an affinity purified rabbit anti-peptide pAb raised to aminoacids 1-14 (MGGKQRDEDDEAYG) of 24P4C12. Anti-24P4C12 immunoreactivebands were visualized by incubation with anti-rabbit-HRP conjugatedsecondary antibody and enhanced chemiluminescence detection. Resultsshow specific recognition of a 90 kD immunoreactive band (arrow) and ahigh molecular weight smear (>132 Kd) that is enhanced by heatdenaturation.

[0033]FIG. 10B. Detection of 24P4C12 protein in 293T cells transfectedwith 24P4C12 cDNA by 24P4C12-specific polyclonal antibodies. Transfected293T cells prepared as for FIG. 10A were lysed in sample buffer andsubjected to strong heat denaturation (100° C.). Western analysis with 2μg/ml of an affinity purified rabbit anti-peptide pAb raised to aminoacids 1-14 (NGGKQRDEDDEAYG) of 24P4C12 was performed as for FIG. 10A.Results show specific recognition of a 90 kD immunoreactive band (arrow)and a high molecular weight smear (>132 Kd) that is enhanced by heatdenaturation.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

[0035] As used herein, the terms “advanced prostate cancer”, “locallyadvanced prostate cancer”, “advanced disease” and “locally advanceddisease” mean prostate cancers that have extended through the prostatecapsule, and are meant to include stage C disease under the AmericanUrological Association (AUA) system, stage C1-C2 disease under theWhitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM(tumor, node, metastasis) system. In general, surgery is not recommendedfor patients with locally advanced disease, and these patients havesubstantially less favorable outcomes compared to patients havingclinically localized (organ-confined) prostate cancer. Locally advanceddisease is clinically identified by palpable evidence of indurationbeyond the lateral border of the prostate, or asymmetry or indurationabove the prostate base. Locally advanced prostate cancer is presentlydiagnosed pathologically following radical prostatectomy if the tumorinvades or penetrates the prostatic capsule, extends into the surgicalmargin, or invades the seminal vesicles.

[0036] As used herein, the terms “metastatic prostate cancer” and“metastatic disease” mean prostate cancers that have spread to regionallymph nodes or to distant sites, and are meant to include stage Ddisease under the AUA system and stage T×N×M+ under the TNM system. Asis the case with locally advanced prostate cancer, surgery is generallynot indicated for patients with metastatic disease, and hormonal(androgen ablation) therapy is the preferred treatment modality.Patients with metastatic prostate cancer eventually develop anandrogen-refractory state within 12 to 18 months of treatmentinitiation, and approximately half of these patients die within 6 monthsthereafter. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are, on balance, characteristicallyosteoblastic rather than osteolytic (i.e., resulting in net boneformation). Bone metastases are found most frequently in the spine,followed by the femur, pelvis, rib cage, skull and humerus. Other commonsites for metastasis include lymph nodes, lung, liver and brain.Metastatic prostate cancer is typically diagnosed by open orlaparoscopic pelvic lymphadenectomy, whole body radionuclide scans,skeletal radiography, and/or bone lesion biopsy.

[0037] As used herein, the term “polynucleotide” means a polymeric formof nucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

[0038] As used herein, the term “polypeptide” means a polymer of atleast 10 amino acids. Throughout the specification, standard threeletter or single letter designations for amino acids are used.

[0039] As used herein, the terms “hybridize”, “hybridizing”,“hybridizes” and the like, used in the context of polynucleotides, aremeant to refer to conventional hybridization conditions, preferably suchas hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, inwhich temperatures for hybridization are above 37 degrees C. andtemperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.,and most preferably to stringent hybridization conditions.

[0040] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0041] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium. citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0042] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and%SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

[0043] In the context of amino acid sequence comparisons, the term“identity” is used to express the percentage of amino acid residues atthe same relative positions that are the same. Also in this context, theterm “homology” is used to express the percentage of amino acid residuesat the same relative positions that are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996): http://blast.wustl/edu/blast/README.html). Furtherdetails regarding amino acid substitutions, which are consideredconservative under such criteria, are provided below.

[0044] Additional definitions are provided throughout the subsectionsthat follow.

[0045] The present invention relates to a novel family of genes andproteins, characterized by multiple transmembrane regions and expressionin prostate cancer. More particularly, the invention provides novelgenes and proteins, designated 24P4C12 and H38087. The invention isbased, in part, on the identification of the 24P4C12 and H38087 genesand on the characterization of the 24P4C12 and H38087 gene expressionpatterns in prostate cancer, normal prostate, and other normal humantissues. As described more fully in the examples that follow, theexpression pattern of the 24P4C12 and H38087 genes was analyzed by: (1)differential expression analysis by RT-PCR using target cDNAs preparedfrom a panel of tissues and cell lines including normal prostate, andthe LAPC-4 AD and AI, and LAPC-9 AD xenografts, (2) tissue specificityanalysis by RT-PCR using cDNAs prepared from 16 normal human tissues,and (3) northern blot analysis of normal prostate and prostate cancerxenograft samples. This combined expression analysis was designed toprovide information on differential expression between AD and AI tissue,clinical prostate cancer and normal prostate, and tissue specificity. Inaddition, initial biological characterization of the 24P4C12 and H38087gene products was undertaken by comparative sequence analysis.

[0046] Nucleotide probes corresponding to all or part of the 24P4C12 andH38087 cDNAs and gene sequences disclosed herein are provided and may beused to isolate or identify other cDNAs encoding all or part of the24P4C12 and H38087 gene sequences. The invention further providedprimers capable of specifically amplifying the 24P4C12 and H38087 genesor their RNA transcripts, and for differentiating between 24P4C12 andH38087 molecules. The invention further provides isolatedpolynucleotides containing coding sequences of the 24P4C12 and H38087gene product(s). Such polynucleotides may be used to express 24P4C12 andH38087 encoded proteins and peptides having a number of further uses.24P4C12 and H38087 gene probes and primers may also be used to detectthe presence or absence of 24P4C12 and H38087 mRNA in various biologicalsamples, for detecting prostate cancer cells and other cells expressing24P4C12 and H38087, and in molecular diagnostic and prognostic assaysfor prostate cancer. Polynucleotides corresponding or complementary tothe 24P4C12 gene may be useful in methods for treating prostate cancer,such as, for example, in modulating or inhibiting 24P4C12 biologicalactivity.

[0047] The invention also provides 24P4C12 and H38087 proteins andpolypeptides that may be used, for example, to generate antibodies.Antibodies capable of specifically binding to and identifying 24P4C12and H38087 proteins or polypeptides may be used to detect the expressionof 24P4C12 and H38087, determine their subcellular location, detect andimage prostate cancer cells and prostate tumors, and modulate or inhibit24P4C12 and H38087 biological activity. These and other aspects of theinvention are described in greater detail in the subsections thatfollow.

[0048] Structure and Expression of 24P4C12

[0049] As is further described in the Examples that follow, the 24P4C12genes and proteins have been characterized using a number of analyticalapproaches. For example, analyses of nucleotide coding and amino acidsequences were conducted in order to identify potentially relatedmolecules, as well as recognizable structural domains, topologicalfeatures, and other elements within the 24P4C12 mRNA and proteinstructures. Northern blot analyses of 24P4C12 mRNA expression wasconducted in order to establish the range of normal and canceroustissues expressing 24P4C12 message.

[0050] The nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO:2) sequences of an approximately 3 kb 24P4C12 combined cDNA sequence areprovided in FIGS. 1A-1D. This 2587 nucleotide sequence encodes a proteinof 710 amino acids, which contains 13 putative transmembrane domains(underlined in FIGS. 1A-1D, and numbered therein as 105-173, 261-329,439-506, 678-746, 768-836, 924-992, 1074-1142, 1245-1313, 1344-1412,1506-1575, 1694-1763, 1803-1871, and 1935-2000). Comparative sequenceanalysis identified two known genes with significant homology to the24P4C12 cDNA sequence, the recently identified murine NG22 gene and theC. elegans gene designated CEESB82F. Both of these genes encode proteinscontaining 12 transmembrane domains. The murine NG22 gene FIGS. 4A-4B;SEQ ID NO: 5) was recently identified as one of many ORFs within agenomic BAC clone that encompasses the MHC class III in the mousegenome.

[0051] Northern blot analysis using an 24P4C12 SSH fragment probeperformed on 16 normal tissues showed expression primarily in prostateand colon, with lower expression detected in kidney, and significantlylower expression detected in pancreas, lung and placenta (FIGS. 2A-2C,3A-3B). To analyze 24P4C12 expression in cancer tissues northernblotting was performed on RNA derived from the LAPC xenografts, andseveral prostate and non-prostate cancer cell lines. The results showhigh expression levels of 24P4C12 in LAPC-4 AD, LAPC-4 AI, LAPC-9 AD,LNCaP and LAPC-4 cell line (FIGS. 2A, 3C, 5). Very high levels aredetected in LAPC-3 AI (FIG. 5). Lower levels are detected in LAPC-9 AI(FIG. 3C). More detailed analysis of the xenografts shows that 24P4C12is highly expressed in the xenografts even when grown within the tibiaof mice (FIG. 5). Northern analysis also shows that 24P4C12 is expressedin the normal prostate and prostate tumor tissues derived from prostatecancer patients (FIG. 6A). These results suggest that 24P4C12 is aprostate gene that is highly expressed in prostate cancer and may have autility as a drug or antibody target in prostate cancer.

[0052] Structure and Expression of H38087

[0053] H38087 was identified as a family member of 24P4C12 by searchingthe dBEST database with the 24P4C12 amino acid sequence using thetblastn tool in NCBI. ESTs that encode protein fragments of homologousproteins were identified. One of these, H38087, was cloned from a testislibrary. The cDNA (clone GTB6) is 2738 bp in size (SEQ ID NO: 6) andencodes a 704 amino acid protein (SEQ ID NO: 7) with 11 putativetransmembrane domains (underlined in FIGS. 7A-7D, and numbered thereinas 152-220, 311-379, 743-811, 830-895, 995-1060, 1133-1201, 1394-1459,1556-1624, 1655-1723, 1859-1924, and 19880-2056). The 58 base pairs of5′ untranslated region are very GC rich (87%), indicating that this genemay contain translational regulatory elements. The amino acid sequencesof 24P4C12 and H38087 are 44% identical and 56% homologous over theentire sequence (FIG. 8).

[0054] Expression analysis shows that H38087 is ubiquitously expressed(FIG. 9), with highest expression levels detected in testis. Expressionis also seen in all the LAPC xenografts. Because H38087 is ubiquitouslyexpressed, it could serve as a control for testing 24P4C12-specifictherapeutics. A 24P4C12-specific therapeutic that affects H38087function could be toxic to normal cells. However, a therapeutic thatselectively affects 24P4C12, but not H38087, may be less toxic to normalcells. Therefore, H38087 protein is useful as a pre-clinical testingtool for therapeutic modalities directed towards 24P4C12.

[0055] Polynucleotides

[0056] One aspect of the invention provides polynucleotidescorresponding or complementary to all or part of a 24P4C12 gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding a 24P4C12 protein and fragments thereof, DNA,RNA, DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 24P4C12 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 24P4C12 gene, mRNA, or to a 24P4C12 encoding polynucleotide(collectively, “24P4C12 polynucleotides”). As used herein, the 24P4C12gene and protein is meant to include the 24P4C12 genes and proteinsspecifically described herein and the genes and proteins correspondingto other 24P4C12 proteins and structurally similar variants of theforegoing. Such other 24P4C12 proteins and variants will generally havecoding sequences that are highly homologous to the 24P4C12 codingsequence, and preferably will share at least about 50% amino acididentity and at least about 60% amino acid homology (using BLASTcriteria), more preferably sharing 70% or greater homology (using BLASTcriteria).

[0057] One embodiment of a 24P4C12 polynucleotide is a 24P4C12polynucleotide having the sequence shown in FIGS. 1A-1D (SEQ ID NO: 1).A 24P4C12 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human 24P4C12 as shown in FIGS. 1A-1D (SEQ ID NO:1), wherein T can also be U; a polynucleotide that encodes all or partof the 24P4C12 protein; a sequence complementary to the foregoing; or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIGS. 1A-1D(SEQ ID NO: 1), from nucleotide residue number 6 through nucleotideresidue number 2138, or having the sequence as shown in FIG. 1E (SEQ IDNO: 3), wherein T can also be U. Another embodiment comprises apolynucleotide encoding a 24P4C12 polypeptide whose sequence is encodedby the cDNA contained in either of the plasmids designated p24P4C12-GTE5or p24P4C12-GTE9 deposited with American Type Culture Collection asDesignation Nos. 207129 and 207084, respectively. Another embodimentcomprises a polynucleotide that is capable of hybridizing understringent hybridization conditions to the human 24P4C12 cDNA shown inFIGS. 1A-1D (SEQ ID NO: 1) or to a polynucleotide fragment thereof.

[0058] Typical embodiments of the invention disclosed herein include24P4C12 polynucleotides encoding specific portions of the 24P4C12 mRNAsequence such as those that encode the protein and fragments thereof.For example, representative embodiments of the invention disclosedherein include: polynucleotides encoding about amino acid 1 to aboutamino acid 10 of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO:2), polynucleotides encoding about amino acid 20 to about amino acid 30of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2),polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2), polynucleotidesencoding about amino acid 40 to about amino acid 50 of the 24P4C12protein shown in FIGS. 1A-1D (SEQ ID NO: 2), polynucleotides encodingabout amino acid 50 to about amino acid 60 of the 24P4C12 protein shownin FIGS. 1A-1D (SEQ ID NO: 2), polynucleotides encoding about amino acid60 to about amino acid 70 of the 24P4C12 protein shown in FIGS. 1A-1D(SEQ ID NO: 2), polynucleotides encoding about amino acid 70 to aboutamino acid 80 of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO:2), polynucleotides encoding about amino acid 80 to about amino acid 90of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2) andpolynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2), etc. Followingthis scheme, polynucleotides (of at least 10 amino acids) encodingportions of the amino acid sequence of amino acids 100-710 of the24P4C12 protein are typical embodiments of the invention.Polynucleotides encoding larger portions of the 24P4C12 protein are alsocontemplated. For example polynucleotides encoding from about amino acid1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50etc.) of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2) may begenerated by a variety of techniques well known in the art.

[0059] Additional illustrative embodiments of the invention disclosedherein include 24P4C12 polynucleotide fragments encoding one or more ofthe biological motifs contained within the 24P4C12 protein sequence. Inone embodiment, typical polynucleotide fragments of the invention canencode one or more of the transmembrane domains disclosed herein. Inanother embodiment, typical polynucleotide fragments of the inventioncan encode one or more of the regions of 24P4C12 that exhibit homologyto H38087, NG22 or CEESB82F. In another embodiment of the invention,typical polynucleotide fragments can encode one or more of the 24P4C12N-glycosylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase phosphorylation, N-myristoylation, oramidation sites, or the leucine zipper pattern, as disclosed in greaterdetail in the text discussing the 24P4C12 protein and polypeptidesbelow. In yet another embodiment of the invention, typicalpolynucleotide fragments can encode sequences that are unique to one ormore 24P4C12 alternative splicing variants.

[0060] The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. For example, as 24P4C12 is shown to bespecifically expressed in prostate cancers (FIGS. 2A, 3C, 5, 6), thesepolynucleotides may be used in methods for assessing the status of24P4C12 gene products in normal versus cancerous tissues. Typically,polynucleotides encoding specific regions of the 24P4C12 protein may beused to assess the presence of perturbations (such as deletions,insertions, point mutations etc.) in specific regions (such regionscontaining a transmembrane domain) of the 24P4C12 gene products.Exemplary assays include both RT-PCR assays as well as single-strandconformation polymorphism (SSCP) analysis (see e.g. Marrogi et al., J.Cutan. Pathol. 26(8): 369-378 (1999), both of which utilizepolynucleotides encoding specific regions of a protein to examine theseregions within the protein.

[0061] Likewise, the invention additionally provides polynucleotidescorresponding or complementary to all or part of a H38087 gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding a H38087 protein and fragments thereof, DNA,RNA, DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a H38087 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa H38087 gene, mRNA, or to a H38087 encoding polynucleotide(collectively, “H38087 polynucleotides”). As used herein, the H38087gene and protein is meant to include the H38087 genes and proteinsspecifically described herein and the genes and proteins correspondingto other H38087 proteins and structurally similar variants of theforegoing. Such other H38087 proteins and variants will generally havecoding sequences that are highly homologous to the H38087 codingsequence, and preferably will share at least about 50% amino acididentity and at least about 60% amino acid homology (using BLASTcriteria), more preferably sharing 70% or greater homology (using BLASTcriteria).

[0062] One embodiment of a H38087 polynucleotide is a H38087polynucleotide having the sequence shown in FIGS. 7A-7D (SEQ ID NO: 6).A H38087 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human H38087 as shown in FIGS. 7A-7D (SEQ ID NO:6), wherein T can also be U; a polynucleotide that encodes all or partof the H38087 protein; a sequence complementary to the foregoing; or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIGS. 7A-7D(SEQ ID NO: 6), from nucleotide residue number 59 through nucleotideresidue number 2173 (using the numbering shown in FIGS. 7A-7D; SEQ IDNO: 6). Another embodiment comprises a polynucleotide that is capable ofhybridizing under stringent hybridization conditions to the human H38087cDNA shown in FIGS. 7A-7D or to a polynucleotide fragment thereof.

[0063] Typical embodiments of the invention disclosed herein includeH38087 polynucleotides encoding specific portions of the H38087 mRNAsequence such as those that encode the protein and fragments thereof.For example, representative embodiments of the invention disclosedherein include: polynucleotides encoding about amino acid 1 to aboutamino acid 10 of the H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7),polynucleotides encoding about amino acid 20 to about amino acid 30 ofthe H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polynucleotidesencoding about amino acid 30 to about amino acid 40 of the H38087protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polynucleotides encodingabout amino acid 40 to about amino acid 50 of the H38087 protein shownin FIGS. 7A-7D (SEQ ID NO: 7), polynucleotides encoding about amino acid50 to about amino acid 60 of the H38087 protein shown in FIGS. 7A-7D(SEQ ID NO: 7), polynucleotides encoding about amino acid 60 to aboutamino acid 70 of the H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7),polynucleotides encoding about amino acid 70 to about amino acid 80 ofthe H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polynucleotidesencoding about amino acid 80 to about amino acid 90 of the H38087protein shown in FIGS. 7A-7D (SEQ ID NO: 7) and polynucleotides encodingabout amino acid 90 to about amino acid 100 of the H38087 protein shownin FIGS. 7A-7D (SEQ ID NO: 7), etc. Following this scheme,polynucleotides (of at least 10 amino acids) encoding portions of theamino acid sequence of amino acids 100-704 of the H38087 protein aretypical embodiments of the invention. Polynucleotides encoding largerportions of the H38087 protein are also contemplated. For examplepolynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the H38087protein shown in FIGS. 7A-7D (SEQ ID NO: 7) may be generated by avariety of techniques well known in the art.

[0064] Additional illustrative embodiments of the invention disclosedherein include H38087 polynucleotide fragments encoding one or more ofthe biological motifs contained within the H38087 protein sequence. Inone embodiment, typical polynucleotide fragments of the invention canencode one or more of the transmembrane domains disclosed herein. Inanother embodiment, typical polynucleotide fragments of the inventioncan encode one or more of the regions of H38087 that exhibit homology to24P4C12, NG22 or CEESB82F. In another embodiment of the invention,typical polynucleotide fragments can encode one or more of the H38087N-glycosylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase phosphorylation, or N-myristoylationsites, or the signal sequence, as disclosed in greater detail in thetext discussing the H38087 protein and polypeptides below. In yetanother embodiment of the invention, typical polynucleotide fragmentscan encode sequences that are unique to one or more H38087 alternativesplicing variants.

[0065] Other specifically contemplated embodiments of the inventiondisclosed herein are genomic DNA, cDNAs, ribozymes, and antisensemolecules, as well as nucleic acid molecules based on an alternativebackbone or including alternative bases, whether derived from naturalsources or synthesized. For example, antisense molecules can be RNAs orother molecules, including peptide nucleic acids (PNAs) or non-nucleicacid molecules such as phosphorothioate derivatives, that specificallybind DNA or RNA in a base pair-dependent manner. A skilled artisan canreadily obtain these classes of nucleic acid molecules using the 24P4C12and H38087 polynucleotides and polynucleotide sequences disclosedherein.

[0066] Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,24P4C12 or H38087. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES,Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis1:1-5 (1988). The 24P4C12 and H38087 antisense oligonucleotides of thepresent invention include derivatives such as S-oligonucleotides(phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra),which exhibit enhanced cancer cell growth inhibitory action. S-oligos(nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfurtransfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990),the disclosures of which are fully incorporated by reference herein.

[0067] The 24P4C12 and H38087 antisense oligonucleotides of the presentinvention typically may be RNA or DNA that is complementary to andstably hybridizes with the first 100 N-terminal codons or last 100C-terminal codons of the 24P4C12 or H38087 genome or the correspondingmRNA. While absolute complementarity is not required, high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 24P4C12 orH38087 mRNA and not to mRNA specifying other regulatory subunits ofprotein kinase. Preferably, the 24P4C12 and H38087 antisenseoligonucleotides of the present invention are a 15 to 30-mer fragment ofthe antisense DNA molecule having a sequence that hybridizes to 24P4C12or H38087 mRNA. Optionally, the 24P4C12 or H38087 antisenseoligonucleotide is a 30-mer oligonucleotide that is complementary to aregion in the first 10 N-terminal codons and last 10 C-terminal codonsof 24P4C12 or H38087. Alternatively, the antisense molecules aremodified to employ ribozymes in the inhibition of 24P4C12 or H38087expression. L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515(1996).

[0068] Further specific embodiments of this aspect of the inventioninclude primers and primer pairs, which allow the specific amplificationof the polynucleotides of the invention or of any specific partsthereof, and probes that selectively or specifically hybridize tonucleic acid molecules of the invention or to any part thereof. Probesmay be labeled with a detectable marker, such as, for example, aradioisotope, fluorescent compound, bioluminescent compound, achemiluminescent compound, metal chelator or enzyme. Such probes andprimers can be used to detect the presence of a 24P4C12 or H38087polynucleotide in a sample and as a means for detecting a cellexpressing a 24P4C12 or H38087 protein.

[0069] Examples of such probes include polypeptides comprising all orpart of the human 24P4C12 cDNA sequence shown in FIGS. 1A-1D (SEQ IDNO: 1) or the human H38087 cDNA sequence FIGS. 7A-7D (SEQ ID NO: 6).Examples of primer pairs capable of specifically amplifying 24P4C12 orH38087 mRNAs are also described in the Examples that follow. As will beunderstood by the skilled artisan, a great many different primers andprobes may be prepared based on the sequences provided herein and usedeffectively to amplify and/or detect a 24P4C12 or H38087 mRNA.

[0070] As used herein, a polynucleotide is said to be “isolated” when itis substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 24P4C12 orH38087 gene or that encode polypeptides other than 24P4C12 or H38087gene product or fragments thereof. A skilled artisan can readily employnucleic acid isolation procedures to obtain an isolated 24P4C12 orH38087 polynucleotide.

[0071] The 24P4C12 or H38087 polynucleotides of the invention are usefulfor a variety of purposes, including but not limited to their use asprobes and primers for the amplification and/or detection of the 24P4C12or H38087 genes, mRNAs, or fragments thereof; as reagents for thediagnosis and/or prognosis of prostate cancer and other cancers; ascoding sequences capable of directing the expression of 24P4C12 orH38087 polypeptides; as tools for modulating or inhibiting theexpression of the 24P4C12 or H38087 genes and/or translation of the24P4C12 or H38087 transcripts; and as therapeutic agents.

[0072] Isolation of 24P4C12- and H38087-Encoding Nucleic Acid Molecules

[0073] The 24P4C12 and H38087 cDNA sequences described herein enable theisolation of other polynucleotides encoding 24P4C12 or H38087 geneproduct(s), as well as the isolation of polynucleotides encoding 24P4C12or H38087 gene product homologs, alternatively spliced isoforms, allelicvariants, and mutant forms of the 24P4C12 or H38087 gene product.Various molecular cloning methods that can be employed to isolate fulllength cDNAs encoding a 24P4C12 or H38087 gene are well known (See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2dedition., Cold Spring Harbor Press, New York, 1989; Current Protocols inMolecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). Forexample, lambda phage cloning methodologies may be convenientlyemployed, using commercially available cloning systems (e.g., Lambda ZAPExpress, Stratagene). Phage clones containing 24P4C12 or H38087 genecDNAs may be identified by probing with a labeled 24P4C12 or H38087 cDNAor a fragment thereof. For example, in one embodiment, the 24P4C12 cDNA(FIGS. 1A-1D; SEQ ID NO: 1) or a portion thereof can be synthesized andused as a probe to retrieve overlapping and full length cDNAscorresponding to a 24P4C12 gene. The 24P4C12 gene itself may be isolatedby screening genomic DNA libraries, bacterial artificial chromosomelibraries (BACs), yeast artificial chromosome libraries (YACs), and thelike, with 24P4C12 DNA probes or primers.

[0074] Recombinant DNA Molecules and Host-Vector Systems

[0075] The invention also provides recombinant DNA or RNA moleculescontaining a 24P4C12 or H38087 polynucleotide, including but not limitedto phages, plasmids, phagemids, cosmids, YACs, BACs, as well as variousviral and non-viral vectors well known in the art, and cells transformedor transfected with such recombinant DNA or RNA molecules. As usedherein, a recombinant DNA or RNA molecule is a DNA or RNA molecule thathas been subjected to molecular manipulation in vitro. Methods forgenerating such molecules are well known (see, for example, Sambrook etal, 1989, supra).

[0076] The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 24P4C12 and/or H38087polynucleotide within a suitable prokaryotic or eukaryotic host cell.Examples of suitable eukaryotic host cells include a yeast cell, a plantcell, or an animal cell, such as a mammalian cell or an insect cell(e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell).Examples of suitable mammalian cells include various prostate cancercell lines such LnCaP, PC-3, DU145, LAPC-4, TsuPr1, other transfectableor transducible prostate cancer cell lines, as well as a number ofmammalian cells routinely used for the expression of recombinantproteins (e.g., COS, CHO, 293, 293T cells). More particularly, apolynucleotide comprising the coding sequence of 24P4C12 or H38087 maybe used to generate 24P4C12 or H38087 proteins or fragments thereofusing any number of host-vector systems routinely used and widely knownin the art.

[0077] A wide range of host-vector systems suitable for the expressionof 24P4C12 and H38087 proteins or fragments thereof are available, seefor example, Sambrook et al., 1989, supra; Current Protocols inMolecular Biology, 1995, supra). Preferred vectors for mammalianexpression include but are not limited to pcDNA 3.1 myc-His-tag(Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991,MCB 11:1785). Using these expression vectors, 24P4C12 or H38087 may bepreferably expressed in several prostate cancer and non-prostate celllines, including for example 293, 293T, rat-1, NIH 3T3, PC-3, LNCaP andTsuPr1. The host-vector systems of the invention are useful for theproduction of a 24P4C12 or H38087 protein or fragment thereof. Suchhost-vector systems may be employed to study the functional propertiesof 24P4C12 or H38087 and 24P4C12 or H38087 mutations.

[0078] Recombinant human 24P4C12 or H38087 protein may be produced bymammalian cells transfected with a construct encoding 24P4C12 or H38087.In an illustrative embodiment described in the Examples, 293T cells canbe transfected with an expression plasmid encoding 24P4C12, the 24P4C12protein is expressed in the 293T cells, and the recombinant 24P4C12protein can be isolated using standard purification methods (e.g.,affinity purification using anti-24P4C12 antibodies). In anotherembodiment, also described in the Examples herein, the 24P4C12 codingsequence is subcloned into the retroviral vector pSRαtkneo and used toinfect various mammalian cell lines, such as NIH 3T3, PC3 and LnCaP inorder to establish 24P4C12 expressing cell lines. Various otherexpression systems well known in the art may also be employed.Expression constructs encoding a leader peptide joined in frame to the24P4C12 coding sequence may be used for the generation of a secretedform of recombinant 24P4C12 protein.

[0079] Proteins encoded by the 24P4C12 or H38087 genes, or by fragmentsthereof, will have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 24P4C12 or H38087 geneproduct. Antibodies raised against a 24P4C12 or H38087 protein orfragment thereof may be useful in diagnostic and prognostic assays, andimaging methodologies in the management of human cancers characterizedby expression of 24P4C12 protein, including but not limited to cancer ofthe prostate. Such antibodies may be expressed intracellularly and usedin methods of treating patients with such cancers. Various immunologicalassays useful for the detection of 24P4C12 and H38087 proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting 24P4C12 or H38087expressing cells (e.g., in radioscintigraphic imaging methods). 24P4C12proteins may also be particularly useful in generating cancer vaccines,as further described below.

[0080] 24P4C12 Polypeptides

[0081] Another aspect of the present invention provides 24P4C12 proteinsand polypeptide fragments thereof. The 24P4C12 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins that combine partsof different 24P4C12 proteins or fragments thereof, as well as fusionproteins of a 24P4C12 protein and a heterologous polypeptide are alsoincluded. Such 24P4C12 proteins will be collectively referred to as the24P4C12 proteins, the proteins of the invention, or 24P4C12. As usedherein, the term “24P4C12 polypeptide” refers to a polypeptide fragmentor a 24P4C12 protein of at least 10 amino acids, preferably at least 15amino acids.

[0082] Specific embodiments of 24P4C12 proteins comprise a polypeptidehaving the amino acid sequence of human 24P4C12 as shown in FIGS. 1A-1D(SEQ ID NO: 2). Alternatively, embodiments of 24P4C12 proteins comprisevariant polypeptides having alterations in the amino acid sequence ofhuman 24P4C12 as shown in FIGS. 1A-1D (SEQ ID NO: 2).

[0083] In general, naturally occurring allelic variants of human 24P4C12will share a high degree of structural identity and homology (e.g., 90%or more identity). Typically, allelic variants of the 24P4C12 proteinswill contain conservative amino acid substitutions within the 24P4C12sequences described herein or will contain a substitution of an aminoacid from a corresponding position in a 24P4C12 homologue. One class of24P4C12 allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular 24P4C12 amino acidsequence, but will further contain a radical departure form thesequence, such as a non-conservative substitution, truncation, insertionor frame shift.

[0084] Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

[0085] Embodiments of the invention disclosed herein include a widevariety of art accepted variants of 24P4C12 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.24P4C12 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassettemutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selectionmutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)] or other known techniques can be performed on the cloned DNA toproduce the 24P4C12 variant DNA. Scanning amino acid analysis can alsobe employed to identify one or more amino acids along a contiguoussequence. Among the preferred scanning amino acids are relatively small,neutral amino acids. Such amino acids include alanine, glycine, serine,and cysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions [Creighton, The Proteins, (W. H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

[0086] As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 710 amino acid sequence of the24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of the 24P4C12protein shown in FIGS. 1A-1D (SEQ ID NO: 2), polypeptides consisting ofabout amino acid 30 to about amino acid 40 of the 24P4C12 protein shownin FIGS. 1A-1D (SEQ ID NO: 2), polypeptides consisting of about aminoacid 40 to about amino acid 50 of the 24P4C12 protein shown in FIGS.1A-1D (SEQ ID NO: 2), polypeptides consisting of about amino acid 50 toabout amino acid 60 of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ IDNO: 2), polypeptides consisting of about amino acid 60 to about aminoacid 70 of the 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2),polypeptides consisting of about amino acid 70 to about amino acid 80 ofthe 24P4C12 protein shown in FIGS. 1A-1D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 24P4C12protein shown in FIGS. 1A-1D (SEQ ID NO: 2) and polypeptides consistingof about amino acid 90 to about amino acid 100 of the 24P4C12 proteinshown in FIGS. 1A-1D (SEQ ID NO: 2), etc. Following this scheme,polypeptides consisting of portions of the amino acid sequence of aminoacids 100-710 of the 24P4C12 protein are typical embodiments of theinvention. Polypeptides consisting of larger portions of the 24P4C12protein are also contemplated. For example polypeptides consisting ofabout amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or30, or 40 or 50 etc.) of the 24P4C12 protein shown in FIGS. 1A-1D (SEQID NO: 2) may be generated by a variety of techniques well known in theart.

[0087] Additional illustrative embodiments of the invention disclosedherein include 24P4C12 polypeptides containing the amino acid residuesof one or more of the biological motifs contained within the 24P4C12polypeptide sequence as shown in FIG. 1A (SEQ ID NO: 2). In oneembodiment, typical polypeptides of the invention can contain one ormore of the transmembrane regions shown in FIGS. 1A-1D (SEQ ID NO: 2),or one or more of the regions of 24P4C12 that exhibit homology toH38087, NG22 OR CEESB82F. In another embodiment, typical polypeptides ofthe invention can contain one or more of the 24P4C12 N-glycosylationsites such as NRSC (SEQ ID NO: 8) at residues 29-32 (numbering fromfirst amino acid residue shown in FIG. 1A), NSTG (SEQ ID NO: 9) atresidues 69-72, NMTV (SEQ ID NO: 10) at residues 155-158, NDTT (SEQ IDNO: 11) at residues 197-200, NLSA (SEQ ID NO: 12) at residues 298-301,NISS (SEQ ID NO: 13) at residues 393-396, NTSC (SEQ ID NO: 14) atresidues 405-408, NSSC (SEQ ID NO: 15) at residues 416-419, and/or NGSL(SEQ ID NO: 16) at residues 678-681. In another embodiment, typicalpolypeptides of the invention can contain one or more of the 24P4C12protein kinase C phosphorylation sites such as SFR at residues 22-24,SVK at residues 218-220, SSK at residues 430-432, TLR at residues494-496, SAK at residues 573-575, and/or SGR at residues 619-621. Inanother embodiment, typical polypeptides of the invention can containone or more of the 24P4C12 casein kinase II phosphorylation sites suchas SCTD (SEQ ID NO: 17) at residues 31-34, SVAE (SEQ ID NO: 18) atresidues 102-105, SCPE (SEQ ID NO: 19) at residues 119-122, TVGE (SEQ IDNO: 20) at residues 135-138, and/or SVQE (SEQ ID NO: 21) at residues304-307. In another embodiment, typical polypeptides of the inventioncan contain one or more of the tyrosine kinase phosphorylation sitessuch as RDEDDEAY (SEQ ID NO: 22) at residues 6-13. In anotherembodiment, typical polypeptides of the invention can contain one ormore of the N-myristoylation sites such as GAYCGM (SEQ ID NO: 23) atresidues 72-77, GMGENK (SEQ ID NO: 24) at residues 76-81, GVPWNM (SEQ IDNO: 25) at residues 151-156, GLIDSL (SEQ ID NO: 26) at residues 207-212,GIYYCW (SEQ ID NO: 27) at residues 272-277, GASISQ (SEQ ID NO: 28) atresidues 287-292, GQMMST (SEQ ID NO: 29) at residues 379-354, GLFWTL(SEQ ID NO: 30) at residues 449-454, and/or GAFASF (SEQ ID NO: 31) atresidues 467-472. In another embodiment, typical polypeptides of theinvention can contain one or more of the amidation sites such as LGKK(SEQ ID NO: 32) at residues 695-698. In another embodiment, typicalpolypeptides of the invention can contain a leucine zipper pattern suchas LFILLLRLVAGPLVLVILGVL (SEQ ID NO: 33) at residues 245-266. Relatedembodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those which contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides.

[0088] In yet another embodiment of the invention, typical polypeptidescan contain amino acid sequences that are unique to one or more 24P4C12alternative splicing variants. The monitoring of alternative splicevariants of 24P4C12 is useful because changes in the alternativesplicing of proteins is suggested as one of the steps in a series ofevents that lead to the progression of cancers (see e.g. Carstens etal., Oncogene 15(250: 3059-3065 (1997)). Consequently, monitoring ofalternative splice variants of 24P4C12 provides an additional means toevaluate syndromes associated with perturbations in 24P4C12 geneproducts such as cancers.

[0089] Polypeptides consisting of one or more of the 24P4C12 motifsdiscussed above are useful in elucidating the specific characteristicsof a malignant phenotype in view of the observation that the 24P4C12motifs discussed above are associated with growth disregulation andbecause 24P4C12 is overexpressed in cancers (FIG. 5). Casein kinase IIand cAMP and cCMP-dependent protein kinase, for example are enzymesknown to be associated with the development of the malignant phenotype(see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon etal., Endocrinology 136(10): 4331-4338 (1995) and Hall et al., NucleicAcids Research 24(6): 1119-1126 (1996)). Moreover, both glycosylationand myristoylation are protein modifications also associated with cancerand cancer progression (see e.g. Dennis et al., Biochim. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)).

[0090] The polypeptides of the preceding paragraphs have a number ofdifferent specific uses. As 24P4C12 is shown to be highly expressed inprostate cancers (FIGS. 2A, 3C, 5, 6), these polypeptides may be used inmethods for assessing the status of 24P4C12 gene products in normalversus cancerous tissues and elucidating the malignant phenotype.Typically, polypeptides encoding specific regions of the 24P4C12 proteinmay be used to assess the presence of perturbations (such as deletions,insertions, point mutations etc.) in specific regions (such as regionscontaining a transmembrane domain) of the 24P4C12 gene products.Exemplary assays can utilize antibodies targeting a 24P4C12 polypeptidescontaining the amino acid residues of one or more of the biologicalmotifs contained within the 24P4C12 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues. Alternatively, 24P4C12 polypeptides containing the amino acidresidues of one or more of the biological motifs contained within the24P4C12 polypeptide sequence can be used to screen for factors thatinteract with that region of 24P4C12.

[0091] As discussed above, redundancy in the genetic code permitsvariation in 24P4C12 gene sequences. In particular, one skilled in theart will recognize specific codon preferences by a specific host speciesand can adapt the disclosed sequence as preferred for a desired host.For example, preferred codon sequences typically have rare codons (i.e.,codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific organism may be calculated, forexample, by utilizing codon usage tables available on the INTERNET atthe following address: http://www.dna.affrc.go.jp/˜nakamura/codon.html.Nucleotide sequences that have been optimized for a particular hostspecies by replacing any codons having a usage frequency of less thanabout 20% are referred to herein as “codon optimized sequences.”

[0092] Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that may be deleterious to gene expression. The GC content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. Where possible, the sequence may also be modified to avoidpredicted hairpin secondary mRNA structures. Other useful modificationsinclude the addition of a translational initiation consensus sequence atthe start of the open reading frame, as described in Kozak, Mol. CellBiol., 9:5073-5080 (1989). Nucleotide sequences that have been optimizedfor expression in a given host species by elimination of spuriouspolyadenylation sequences, elimination of exon/intron splicing signals,elimination of transposon-like repeats and/or optimization of GC contentin addition to codon optimization are referred to herein as an“expression enhanced sequence.”

[0093] 24P4C12 proteins may be embodied in many forms, preferably inisolated form. As used herein, a protein is said to be “isolated” whenphysical, mechanical or chemical methods are employed to remove the24P4C12 protein from cellular constituents that are normally associatedwith the protein. A skilled artisan can readily employ standardpurification methods to obtain an isolated 24P4C12 protein. A purified24P4C12 protein molecule will be substantially free of other proteins ormolecules that impair the binding of 24P4C12 to antibody or otherligand. The nature and degree of isolation and purification will dependon the intended use. Embodiments of a 24P4C12 protein include a purified24P4C12 protein and a functional, soluble 24P4C12 protein. In one form,such functional, soluble 24P4C12 proteins or fragments thereof retainthe ability to bind antibody or other ligand.

[0094] The invention also provides 24P4C12 polypeptides comprisingbiologically active fragments of the 24P4C12 amino acid sequence, suchas a polypeptide corresponding to part of the amino acid sequence for24P4C12 as shown in FIGS. 1A-1D (SEQ ID NO: 2). Such polypeptides of theinvention exhibit properties of the 24P4C12 protein, such as the abilityto elicit the generation of antibodies that specifically bind an epitopeassociated with the 24P4C12 protein. 24P4C12 polypeptides can begenerated using standard peptide synthesis technology or using chemicalcleavage methods well known in the art based on the amino acid sequencesof the human 24P4C12 proteins disclosed herein. Alternatively,recombinant methods can be used to generate nucleic acid molecules thatencode a polypeptide fragment of a 24P4C12 protein. In this regard, the24P4C12-encoding nucleic acid molecules described herein provide meansfor generating defined fragments of 24P4C12 proteins. 24P4C12polypeptides are particularly useful in generating and characterizingdomain specific antibodies (e.g., antibodies recognizing anextracellular or intracellular epitope of a 24P4C12 protein), inidentifying agents or cellular factors that bind to 24P4C12 or aparticular structural domain thereof, and in various therapeuticcontexts, including but not limited to cancer vaccines.

[0095] 24P4C12 polypeptides containing particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-24P4C12 antibodies or in identifying cellular factors thatbind to 24P4C12.

[0096] In an embodiment described in the examples that follow, 24P4C12can be conveniently expressed in cells (such as 293T cells) transfectedwith a commercially available expression vector such as a CMV-drivenexpression vector encoding 24P4C12 with a C-terminal 6× His and MYC tag(pcDNA3.1/mycHIS, Invitrogen). The HIS-tagged 24P4C12 expressed in cellsmay be purified using a nickel column using standard techniques.

[0097] Modifications of 24P4C12 such as covalent modifications areincluded within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 24P4C12polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe 24P4C12. Another type of covalent modification of the 24P4C12polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequence24P4C12 (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence 24P4C12. In addition, the phrase includes qualitativechanges in the glycosylation of the native proteins, involving a changein the nature and proportions of the various carbohydrate moietiespresent. Another type of covalent modification of 24P4C12 compriseslinking the 24P4C12 polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0098] The 24P4C12 of the present invention may also be modified in away to form a chimeric molecule comprising 24P4C12 fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the 24P4C12 with apolyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 24P4C12. In analternative embodiment, the chimeric molecule may comprise a fusion ofthe 24P4C12 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 24P4C12 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgGI molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0099] H38087 Polypeptides

[0100] Another aspect of the present invention provides H38087 proteinsand polypeptide fragments thereof. The H38087 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein. Fusion proteins that combineparts of different H38087 proteins or fragments thereof, as well asfusion proteins of a H38087 protein and a heterologous polypeptide arealso included. Such H38087 proteins will be collectively referred to asthe H38087 proteins or H38087. As used herein, the term “H38087polypeptide” refers to a polypeptide fragment or a H38087 protein of atleast 10 amino acids, preferably at least 15 amino acids.

[0101] As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 704 amino acid sequence of theH38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of the H38087protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polypeptides consisting ofabout amino acid 30 to about amino acid 40 of the H38087 protein shownin FIGS. 7A-7D (SEQ ID NO: 7), polypeptides consisting of about aminoacid 40 to about amino acid 50 of the H38087 protein shown in FIGS.7A-7D (SEQ ID NO: 7), polypeptides consisting of about amino acid 50 toabout amino acid 60 of the H38087 protein shown in FIGS. 7A-7D (SEQ IDNO: 7), polypeptides consisting of about amino acid 60 to about aminoacid 70 of the H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7),polypeptides consisting of about amino acid 70 to about amino acid 80 ofthe H38087 protein shown in FIGS. 7A-7D (SEQ ID NO: 7), polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the H38087protein shown in FIGS. 7A-7D (SEQ ID NO: 7) and polypeptides consistingof about amino acid 90 to about amino acid 100 of the H38087 proteinshown in FIGS. 7A-7D (SEQ ID NO: 7), etc. Following this scheme,polypeptides consisting of portions of the amino acid sequence of aminoacids 100-710 of the H38087 protein are typical embodiments of theinvention. Polypeptides consisting of larger portions of the H38087protein are also contemplated. For example polypeptides consisting ofabout amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or30, or 40 or 50 etc.) of the H38087 protein shown in FIGS. 7A-7D (SEQ IDNO: 7) may be generated by a variety of techniques well known in theart.

[0102] Additional illustrative embodiments of the invention disclosedherein include H38087 polypeptides containing the amino acid residues ofone or more of the biological motifs contained within the H38087polypeptide sequence as shown in FIG. 7 (SEQ ID NO: 7). In oneembodiment, typical polypeptides of the invention can contain one ormore of the transmembrane regions shown in FIGS. 7A-7D, or one or moreof the regions of H38087 that exhibit homology to 24P4C12, NG22 ORCEESB82F. In another embodiment, typical polypeptides of the inventioncan contain one or more of the H38087 N-glycosylation sites such as NETT(SEQ ID NO: 46) at residues 185-188 (numbering from first amino acidresidue shown in FIG. 7), NITD (SEQ ID NO: 47) at residues 198-201,and/or NKTN (SEQ ID NO: 48) at residues 695-698. In another embodiment,typical polypeptides of the invention can contain one or more of theH38087 protein kinase C phosphorylation sites such as TFK at residues19-21, SSR at residues 126-128, SRK at residues 195-197, TAK at residues402-404, SAR at residues 574-576, THR at residues 620-622, TLK atresidues 689-691, and/or TNK at residues 697-699. In another embodiment,typical polypeptides of the invention can contain one or more of theH38087 casein kinase II phosphorylation sites such as THGD (SEQ ID NO:49) at residues 54-57, SRGE (SEQ ID NO: 50) at residues 67-70, TKNE (SEQID NO: 51) at residues 77-80, SSRD (SEQ ID NO: 52) at residues 126-129,TTYE (SEQ ID NO: 53) at residues 187-190, TYED (SEQ ID NO: 54) atresidues 188-191, SLVD (SEQ ID NO: 55) at residues 293-296, SILE (SEQ IDNO: 56) at residues 321-234, TSNE (SEQ ID NO: 57) at residues 385-388and/or SSHE (SEQ ID NO: 58) at residues 413-416. In another embodiment,typical polypeptides of the invention can contain one or more of thetyrosine kinase phosphorylation sites such as RSSRDFEYY (SEQ ID NO: 59)at residues 125-133. In another embodiment, typical polypeptides of theinvention can contain one or more of the N-myristoylation sites such asGQKGTK (SEQ ID NO: 60) at residues 73-78, GNETTY (SEQ ID NO: 61) atresidues 184-189, GSRKNI (SEQ ID NO: 62) at residues 194-199, GAKKAN(SEQ ID NO: 63) at residues 205-210, GVLEAR (SEQ ID NO: 64) at residues211-216, GLVIAM (SEQ ID NO: 65) at residues 236-241, GIFHCY (SEQ ID NO:66) at residues 273-278, GSDVSL (SEQ ID NO: 67) at residues 289-294,GGESGY (SEQ ID NO: 68) at residues 431-436, GAFASY (SEQ ID NO: 69) atresidues 468-473, and/or GTNFCT (SEQ ID NO: 70) at residues 568-573.Related embodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those which contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides.

[0103] The H38087 polypeptides of the invention can be modified,generated and used in manners analogous to those described above for24P4C12 polypeptides, as would be known and appreciated by those skilledin the art.

[0104] 24P4C12 Antibodies

[0105] The term “antibody” is used in the broadest sense andspecifically covers single anti-24P4C12 monoclonal antibodies (includingagonist, antagonist and neutralizing antibodies) and anti-24P4C12antibody compositions with polyepitopic specificity. The term“monoclonal antibody” (mAb) as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the antibodies comprising the individual population are identical exceptfor possible naturally-occurring mutations that may be present in minoramounts.

[0106] Another aspect of the invention provides antibodies that bind to24P4C12 proteins and polypeptides. The most preferred antibodies willspecifically bind to a 24P4C12 protein and will not bind (or will bindweakly) to non-24P4C12 proteins and polypeptides. Anti-24P4C12antibodies that are particularly contemplated include monoclonal andpolyclonal antibodies as well as fragments containing the antigenbinding domain and/or one or more complementarity determining regions ofthese antibodies. As used herein, an antibody fragment is defined as atleast a portion of the variable region of the immunoglobulin moleculethat binds to its target, i.e., the antigen binding region.

[0107] 24P4C12 antibodies of the invention may be particularly useful inprostate cancer diagnostic and prognostic assays, and imagingmethodologies. Intracellularly expressed antibodies (e.g., single chainantibodies) may be therapeutically useful in treating cancers in whichthe expression of 24P4C12 is involved, such as for example advanced andmetastatic prostate cancers. Also useful in therapeutic methods fortreatment of prostate cancer are systemically administered 24P4C12antibodies that interfere with 24P4C12 function or that targetextracellular regions of 24P4C12 for delivery of a toxin or therapeuticmolecule. Such delivery of a toxin or therapeutic molecule can beachieved using known methods of conjugating a second molecule to the24P4C12 antibody or fragment thereof. Similarly, such antibodies may beuseful in the treatment, diagnosis, and/or prognosis of other cancers,to the extent 24P4C12 is also expressed or overexpressed in other typesof cancer.

[0108] The invention also provides various immunological assays usefulfor the detection and quantification of 24P4C12 and mutant 24P4C12proteins and polypeptides. Such assays generally comprise one or more24P4C12 antibodies capable of recognizing and binding a 24P4C12 ormutant 24P4C12 protein, as appropriate, and may be performed withinvarious immunological assay formats well known in the art, including butnot limited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer and other cancers expressing24P4C12 are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled 24P4C12 antibodies.Such assays may be clinically useful in the detection, monitoring, andprognosis of 24P4C12 expressing cancers, such as prostate cancer.

[0109] 24P4C12 antibodies may also be used in methods for purifying24P4C12 and mutant 24P4C12 proteins and polypeptides and for isolating24P4C12 homologues and related molecules. For example, in oneembodiment, the method of purifying a 24P4C12 protein comprisesincubating a 24P4C12 antibody, which has been coupled to a solid matrix,with a lysate or other solution containing 24P4C12 under conditions thatpermit the 24P4C12 antibody to bind to 24P4C12; washing the solid matrixto eliminate impurities; and eluting the 24P4C12 from the coupledantibody. Other uses of the 24P4C12 antibodies of the invention includegenerating anti-idiotypic antibodies that mimic the 24P4C12 protein.

[0110] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies may be prepared by immunizing asuitable mammalian host using a 24P4C12 protein, peptide, or fragment,in isolated or immunoconjugated form (Antibodies: A Laboratory Manual,CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, ColdSpring Harbor Press, NY (1989)). In addition, fusion proteins of 24P4C12may also be used, such as a 24P4C12 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the openreading frame amino acid sequence of FIGS. 1A-1D (SEQ ID NO: 2) may beproduced and used as an immunogen to generate appropriate antibodies. Inanother embodiment, a 24P4C12 peptide may be synthesized and used as animmunogen.

[0111] In addition, naked DNA immunization techniques known in the artmay be used (with or without purified 24P4C12 protein or 24P4C12expressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0112] The amino acid sequence of the 24P4C12 as shown in FIGS. 1A-1D(SEQ ID NO: 2) may be used to select specific regions of the 24P4C12protein for generating antibodies. For example, hydrophobicity andhydrophilicity analyses of the 24P4C12 amino acid sequence may be usedto identify hydrophilic regions in the 24P4C12 structure. Regions of the24P4C12 protein that show immunogenic structure, as well as otherregions and domains, can readily be identified using various othermethods known in the art, such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Methods for the generation of 24P4C12 antibodies are further illustratedby way of the examples provided herein.

[0113] Methods for preparing a protein or polypeptide for use as animmunogen and for preparing immunogenic conjugates of a protein with acarrier such as BSA, KLH, or other carrier proteins are well known inthe art. In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a 24P4C12 immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

[0114] Polyclonal 24P4C12 antibodies can be prepared using conventionaltechniques known in the art. A representative protocol for thepreparation of such antibodies is described in the Examples that follow.Polyclonal antibodies can be useful for sensitive detection of multipleepitopes associated with 24P4C12.

[0115] 24P4C12 monoclonal antibodies may be produced by various meanswell known in the art. For example, immortalized cell lines that secretea desired monoclonal antibody may be prepared using the standardhybridoma technology of Kohler and Milstein or modifications thatimmortalize producing B cells, as is generally known. The immortalizedcell lines secreting the desired antibodies are screened by immunoassayin which the antigen is the 24P4C12 protein or a 24P4C12 fragment. Whenthe appropriate immortalized cell culture secreting the desired antibodyis identified, the cells may be expanded and antibodies produced eitherfrom in vitro cultures or from ascites fluid.

[0116] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 24P4C12 protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human 24P4C12 antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522-525;Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988,Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl.Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539).

[0117] Fully human 24P4C12 monoclonal antibodies may be generated usingcloning technologies employing large human Ig gene combinatoriallibraries (i.e., phage display) (Griffiths and Hoogenboom, Building anin vitro immune system: human antibodies from phage display libraries.In: Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human 24P4C12 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

[0118] Reactivity of 24P4C12 antibodies with a 24P4C12 protein may beestablished by a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,24P4C12 proteins, peptides, 24P4C12-expressing cells or extractsthereof.

[0119] A 24P4C12 antibody or fragment thereof of the invention may belabeled with a detectable marker or conjugated to a second molecule.Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a metal chelator or an enzyme. A secondmolecule for conjugation to the 24P4C12 antibody can be selected inaccordance with the intended use. For example, for therapeutic use, thesecond molecule can be a toxin or therapeutic agent. Further,bi-specific antibodies specific for two or more 24P4C12 epitopes may begenerated using methods generally known in the art. Homodimericantibodies may also be generated by cross-linking techniques known inthe art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

[0120] H38087 Antibodies

[0121] The invention also provides antibodies, both polyclonal andmonoclonal, directed against H38087. These antibodies can be modified,generated and used in manners analogous to those described above for24P4C12 antibodies. The ubiquitous expression of H38087, however, makesit likely to be useful as a control for testing 24P4C12-specifictherapeutics, and possibly for comparison in 24P4C12 diagnosticapplications. A 24P4C12-specific therapeutic that affects H38087function could be toxic to normal cells. However, a therapeutic thatselectively affects 24P4C12, but not H38087, may be less toxic to normalcells. Therefore, H38087 proteins and antibodies can be useful aspre-clinical testing tools for therapeutic modalities directed towards24P4C12.

[0122] 24P4C12 Transgenic Animals

[0123] Nucleic acids that encode 24P4C12 or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding 24P4C12 can be used to clonegenomic DNA encoding 24P4C12 in accordance with established techniquesand the genomic sequences used to generate transgenic animals thatcontain cells that express DNA encoding 24P4C12. Methods for generatingtransgenic animals, particularly animals such as mice or rats, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for 24P4C12 transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding 24P4C12 introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding 24P4C12. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0124] Alternatively, non-human homologues of 24P4C12 can be used toconstruct a 24P4C12 “knock out” animal that has a defective or alteredgene encoding 24P4C12 as a result of homologous recombination betweenthe endogenous gene encoding 24P4C12 and altered genomic DNA encoding24P4C12 introduced into an embryonic cell of the animal. For example,cDNA encoding 24P4C12 can be used to clone genomic DNA encoding 24P4C12in accordance with established techniques. A portion of the genomic DNAencoding 24P4C12 can be deleted or replaced with another gene, such as agene encoding a selectable marker that can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the 24P4C12 polypeptide.

[0125] Likewise, H38087 transgenic animals can be prepared using nucleicacids that encode H38087.

[0126] Methods for the Detection of 24P4C12

[0127] Another aspect of the present invention relates to methods fordetecting 24P4C12 polynucleotides and 24P4C12 proteins and variantsthereof, as well as methods for identifying a cell that expresses24P4C12. 24P4C12 appears to be expressed in the LAPC xenografts that arederived from lymph-node and bone metastasis of prostate cancer and theexpression profile of 24P4C12 makes it a potential diagnostic marker formetastasized disease. In this context, the status of 24P4C12 geneproducts may provide information useful for predicting a variety offactors including susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. As discussed in detail below,the status of 24P4C12 gene products in patient samples may be analyzedby a variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of Northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

[0128] More particularly, the invention provides assays for thedetection of 24P4C12 polynucleotides in a biological sample, such asserum, bone, prostate, and other tissues, urine, semen, cellpreparations, and the like. Detectable 24P4C12 polynucleotides include,for example, a 24P4C12 gene or fragments thereof, 24P4C12 mRNA,alternative splice variant 24P4C12 mRNAs, and recombinant DNA or RNAmolecules containing a 24P4C12 polynucleotide. A number of methods foramplifying and/or detecting the presence of 24P4C12 polynucleotides arewell known in the art and may be employed in the practice of this aspectof the invention.

[0129] In one embodiment, a method for detecting a 24P4C12 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing a 24P4C12 polynucleotides as sense and antisense primers toamplify 24P4C12 cDNAs therein; and detecting the presence of theamplified 24P4C12 cDNA. Optionally, the sequence of the amplified24P4C12 cDNA can be determined. In another embodiment, a method ofdetecting a 24P4C12 gene in a biological sample comprises firstisolating genomic DNA from the sample; amplifying the isolated genomicDNA using 24P4C12 polynucleotides as sense and antisense primers toamplify the 24P4C12 gene therein; and detecting the presence of theamplified 24P4C12 gene. Any number of appropriate sense and antisenseprobe combinations may be designed from the nucleotide sequencesprovided for the 24P4C12 FIGS. 1A-1D; SEQ ID NO: 1) and used for thispurpose.

[0130] The invention also provides assays for detecting the presence ofa 24P4C12 protein in a tissue or other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a 24P4C12 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a 24P4C12 protein in a biological sample comprises first contactingthe sample with a 24P4C12 antibody, a 24P4C12-reactive fragment thereof,or a recombinant protein containing an antigen binding region of a24P4C12 antibody; and then detecting the binding of 24P4C12 protein inthe sample thereto.

[0131] Methods for identifying a cell that expresses 24P4C12 are alsoprovided. In one embodiment, an assay for identifying a cell thatexpresses a 24P4C12 gene comprises detecting the presence of 24P4C12mRNA in the cell. Methods for the detection of particular mRNAs in cellsare well known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeled24P4C12 riboprobes, northern blot and related techniques) and variousnucleic acid amplification assays (such as RT-PCR using complementaryprimers specific for 24P4C12, and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses a 24P4C12gene comprises detecting the presence of 24P4C12 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of 24P4C12proteins and 24P4C12 expressing cells. 24P4C12 expression analysis mayalso be useful as a tool for identifying and evaluating agents thatmodulate 24P4C12 gene expression. For example, 24P4C12 expression issignificantly upregulated in prostate cancers, and may also be expressedin other cancers. Identification of a molecule or biological agent thatcould inhibit 24P4C12 expression or over-expression in cancer cells maybe of therapeutic value. Such an agent may be identified by using ascreen that quantifies 24P4C12 expression by RT-PCR, nucleic acidhybridization or antibody binding.

[0132] Monitoring the Status of 24P4C12 and its Products

[0133] Assays that evaluate the status of the 24P4C12 gene and 24P4C12gene products in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because 24P4C12 mRNA is so highly expressed in prostate cancersrelative to normal tissues, assays that evaluate the relative levels of24P4C12 mRNA transcripts or proteins in a biological sample may be usedto diagnose a disease associated with 24P4C12 disregulation such ascancer and may provide prognostic information useful in definingappropriate therapeutic options. Similarly, assays that evaluate theintegrity 24P4C12 nucleotide and amino acid sequences in a biologicalsample, may also be used in this context.

[0134] The finding that 24P4C12 mRNA is so highly expressed in prostatecancers, and not in normal tissue, provides evidence that this gene isassociated with disregulated cell growth and therefore identifies thisgene and its products as targets that the skilled artisan can use toevaluate biological samples from individuals suspected of having adisease associated with 24P4C12 disregulation. In this context, theevaluation of the expression status of 24P4C12 gene and its products canbe used to gain information on the disease potential of a tissue sample.The terms “expression status” in this context is used to broadly referto the variety of factors involved in the expression, function andregulation of a gene and its products such as the level of mRNAexpression, the integrity of the expressed gene products (such as thenucleic and amino acid sequences) and transcriptional and translationalmodifications to these molecules.

[0135] The expression status of 24P4C12 may provide information usefulfor predicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining 24P4C12 expression status and diagnosing cancers thatexpress 24P4C12, such as cancers of the prostate, breast, bladder, lung,bone, colon, pancreatic, testicular, cervical and ovarian cancers.24P4C12 expression status in patient samples may be analyzed by a numberof means well known in the art, including without limitation,immunohistochemical analysis, in situ hybridization, RT-PCR analysis onlaser capture micro-dissected samples, western blot analysis of clinicalsamples and cell lines, and tissue array analysis. Typical protocols forevaluating the expression status of the 24P4C12 gene and gene productscan be found, for example in Current Protocols In Molecular Biology,Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting]and 18 [PCR Analysis], Frederick M. Ausubul et al. eds., 1995.

[0136] In one aspect, the invention provides methods for monitoring24P4C12 gene products by determining the status of 24P4C12 gene productsexpressed by cells in a test tissue sample from an individual suspectedof having a disease associated with disregulated cell growth (such ashyperplasia or cancer) and then comparing the status so determined tothe status of 24P4C12 gene products in a corresponding normal sample,the presence of aberrant 24P4C12 gene products in the test samplerelative to the normal sample providing an indication of the presence ofdisregulated cell growth within the cells of the individual.

[0137] In another aspect, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant increase in 24P4C12 mRNA or protein expressionin a test cell or tissue sample relative to expression levels in thecorresponding normal cell or tissue. The presence of significant 24P4C12expression in these tissues may be useful to indicate the emergence,presence and/or severity of cancer.

[0138] In a related embodiment, 24P4C12 expression status may bedetermined at the protein level rather than at the nucleic acid level.For example, such a method or assay would comprise determining the levelof 24P4C12 protein expressed by cells in a test tissue sample andcomparing the level so determined to the level of 24P4C12 expressed in acorresponding normal sample. In one embodiment, the presence of 24P4C12protein is evaluated, for example, using immunohistochemical methods.24P4C12 antibodies or binding partners capable of detecting 24P4C12protein expression may be used in a variety of assay formats well knownin the art for this purpose.

[0139] In other related embodiments, one can evaluate the integrity24P4C12 nucleotide and amino acid sequences in a biological sample inorder to identify perturbations in the structure of these molecules suchas insertions, deletions, substitutions and the like. Such embodimentsare useful because perturbations in the nucleotide and amino acidsequences are observed in a large number of proteins associated with agrowth disregulated phenotype (see e.g. Marrogi et al., J. Cutan.Pathol. 26(8): 369-378 (1999)). In this context, a wide variety ofassays for observing perturbations in nucleotide and amino acidsequences are well known in the art. For example, the size and structureof nucleic acid or amino acid sequences of 24P4C12 gene products may beobserved by the northern, Southern, western, PCR and DNA sequencingprotocols discussed herein. In addition, other methods for observingperturbations in nucleotide and amino acid sequences such as singlestrand conformation polymorphism analysis are well known in the art (seee.g. U.S. Pat. Nos. 5,382,510 and 5,952,170).

[0140] In another related embodiment, the invention provides assaysuseful in determining the presence of cancer in an individual,comprising detecting a significant change in the 24P4C12 alternativesplice variants expressed in a test cell or tissue sample relative toexpression levels in the corresponding normal cell or tissue. Themonitoring of alternative splice variants of 24P4C12 is useful becausechanges in the alternative splicing of proteins is suggested as one ofthe steps in a series of events that lead to the progression of cancers(see e.g. Carstens et al., Oncogene 15(250: 3059-3065 (1997)).

[0141] Gene amplification provides an additional method of assessing thestatus of 24P4C12. Gene amplification may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0142] In addition to the tissues discussed above, peripheral blood maybe conveniently assayed for the presence of cancer cells, including butnot limited to prostate cancers, using RT-PCR to detect 24P4C12expression. The presence of RT-PCR amplifiable 24P4C12 mRNA provides anindication of the presence of the cancer. RT-PCR detection assays fortumor cells in peripheral blood are currently being evaluated for use inthe diagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

[0143] A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting24P4C12 mRNA or 24P4C12 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 24P4C12 mRNAexpression present is proportional to the degree of susceptibility. In aspecific embodiment, the presence of 24P4C12 in prostate tissue isexamined, with the presence of 24P4C12 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In a closely related embodiment, one canevaluate the integrity 24P4C12 nucleotide and amino acid sequences in abiological sample in order to identify perturbations in the structure ofthese molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in 24P4C12 geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

[0144] Yet another related aspect of the invention is directed tomethods for gauging tumor aggressiveness. In one embodiment, a methodfor gauging aggressiveness of a tumor comprises determining the level of24P4C12 mRNA or 24P4C12 protein expressed by cells in a sample of thetumor, comparing the level so determined to the level of 24P4C12 mRNA or24P4C12 protein expressed in a corresponding normal tissue taken fromthe same individual or a normal tissue reference sample, wherein thedegree of 24P4C12 mRNA or 24P4C12 protein expression in the tumor samplerelative to the normal sample indicates the degree of aggressiveness. Ina specific embodiment, aggressiveness of prostate tumors is evaluated bydetermining the extent to which 24P4C12 is expressed in the tumor cells,with higher expression levels indicating more aggressive tumors. In aclosely related embodiment, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, with the presence ofone or more perturbations indicating more aggressive tumors.

[0145] Yet another related aspect of the invention is directed tomethods for observing the progression of a malignancy in an individualover time. In one embodiment, methods for observing the progression of amalignancy in an individual over time comprise determining the level of24P4C12 mRNA or 24P4C12 protein expressed by cells in a sample of thetumor, comparing the level so determined to the level of 24P4C12 mRNA or24P4C12 protein expressed in an equivalent tissue sample taken from thesame individual at a different time, wherein the degree of 24P4C12 mRNAor 24P4C12 protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining the extent towhich 24P4C12 expression in the tumor cells alters over time, withhigher expression levels indicating a progression of the cancer. In aclosely related embodiment, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, with the presence ofone or more perturbations indicating a progression of the cancer.

[0146] The above diagnostic approaches may be combined with any one of awide variety of prognostic and diagnostic protocols known in the art.For example, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof 24P4C12 gene and 24P4C12 gene products (or perturbations in 24P4C12gene and 24P4C12 gene products) and a factor that is associated withmalignancy as a means of diagnosing and prognosticating the status of atissue sample. In this context, a wide variety of factors associatedwith malignancy may be utilized such as the expression of genesotherwise associated with malignancy (including PSA, PSCA and PSMexpression) as well as gross cytological observations (see e.g. Bockinget al., Anal Quant Cytol. 6(2):74-88 (1984); Eptsein, Hum Pathol.February 1995; 26(2):223-9 (1995); Thorson et al., Mod Pathol. June1998; 11(6):543-51; Baisden et al., Am J Surg Pathol. 23(8):918-2491999)). Methods for observing a coincidence between the expression of24P4C12 gene and 24P4C12 gene products (or perturbations in 24P4C12 geneand 24P4C12 gene products) and an additional factor that is associatedwith malignancy are useful, for example, because the presence of a setor constellation of specific factors that coincide provides informationcrucial for diagnosing and prognosticating the status of a tissuesample.

[0147] In a typical embodiment, methods for observing a coincidencebetween the expression of 24P4C12 gene and 24P4C12 gene products (orperturbations in 24P4C12 gene and 24P4C12 gene products) and a factorthat is associated with malignancy entails detecting the overexpressionof 24P4C12 mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample, and observinga coincidence of 24P4C12 mRNA or protein and PSA mRNA or proteinoverexpression. In a specific embodiment, the expression of 24P4C12 andPSA mRNA in prostate tissue is examined. In a preferred embodiment, thecoincidence of 24P4C12 and PSA mRNA overexpression in the sampleprovides an indication of prostate cancer, prostate cancersusceptibility or the emergence or existence of a prostate tumor.

[0148] Methods for detecting and quantifying the expression of 24P4C12mRNA or protein are described herein and use standard nucleic acid andprotein detection and quantification technologies well known in the art.Standard methods for the detection and quantification of 24P4C12 mRNAinclude in situ hybridization using labeled 24P4C12 riboprobes, northernblot and related techniques using 24P4C12 polynucleotide probes, RT-PCRanalysis using primers specific for 24P4C12, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR may beused to detect and quantify 24P4C12 mRNA expression as described in theExamples that follow. Any number of primers capable of amplifying24P4C12 may be used for this purpose, including but not limited to thevarious primer sets specifically described herein. Standard methods forthe detection and quantification of protein may be used for thispurpose. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 24P4C12 protein may be used inan immunohistochemical assay of biopsied tissue.

[0149] Identifying Molecules that Interact with 24P4C12

[0150] The 24P4C12 protein sequences disclosed herein allow the skilledartisan to identify molecules that interact with them via any one of avariety of art accepted protocols. For example one can utilize one ofthe variety of so-called interaction trap systems (also referred to asthe “two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

[0151] Alternatively, one can identify molecules that interact with24P4C12 protein sequences by screening peptide libraries. In suchmethods, peptides that bind to selected receptor molecules such as24P4C12 are identified by screening libraries that encode a random orcontrolled collection of amino acids. Peptides encoded by the librariesare expressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest. Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with 24P4C12 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

[0152] Alternatively, cell lines expressing 24P4C12 can be used toidentify protein-protein interactions mediated by 24P4C12. Thispossibility can be examined using immunoprecipitation techniques asshown by others (Hamilton B J, et al. Biochem. Biophys. Res. Commun.1999, 261:646-51). Typically 24P4C12 protein can be immunoprecipitatedfrom 24P4C12 expressing prostate cancer cell lines using anti-24P4C12antibodies. Alternatively, antibodies against His-tag can be used in acell line engineered to express 24P4C12 (using, e.g., vectors mentionedabove). The immuno-precipitated complex can be examined for proteinassociation by procedures such as western blotting, ³⁵S-methioninelabeling of proteins, protein microsequencing, silver staining and twodimensional gel electrophoresis.

[0153] Related embodiments of such screening assays include methods foridentifying small molecules that interact with 24P4C12. Typical methodsare discussed for example in U.S. Pat. No. 5,928,868 and include methodsfor forming hybrid ligands in which at least one ligand is a smallmolecule. In an illustrative embodiment, the hybrid ligand is introducedinto cells that in turn contain a first and a second expression vector.Each expression vector includes DNA for expressing a hybrid protein thatencodes a target protein linked to a coding sequence for atranscriptional module. Each cell further contains a reporter gene, theexpression of which is conditioned on the proximity of the first andsecond hybrid proteins to each other, an event that occurs only if thehybrid ligand binds to target sites on both hybrid proteins. Those cellsthat express the reporter gene are selected and the unknown smallmolecule or the unknown hybrid protein is identified.

[0154] A typical embodiment of this invention consists of a method ofscreening for a molecule that interacts with a 24P4C12 amino acidsequence shown in FIGS. 1A-1D, comprising the steps of contacting apopulation of molecules with the 24P4C12 amino acid sequence, allowingthe population of molecules and the 24P4C12 amino acid sequence tointeract under conditions that facilitate an interaction, determiningthe presence of a molecule that interacts with the 24P4C12 amino acidsequence and then separating molecules that do not interact with the24P4C12 amino acid sequence from molecules that do interact with the24P4C12 amino acid sequence. In a specific embodiment, the methodfurther includes purifying a molecule that interacts with the 24P4C12amino acid sequence. In a preferred embodiment, the 24P4C12 amino acidsequence is contacted with a library of peptides.

[0155] Therapeutic Methods and Compositions

[0156] The identification of 24P4C12 as a prostate tumor-associatedprotein, opens a number of therapeutic approaches to the treatment ofsuch cancers. As discussed above, it is possible that 24P4C12 functionsas a receptor involved in activating or modulating proliferationsignals, and that it presents epitopes at the cell surface that can betargeted for therapy.

[0157] The expression profile of 24P4C12 is reminiscent of the MAGEs,PSA and PMSA, which are tissue-specific genes that are up-regulated inmelanomas and other cancers (Van den Eynde and Boon, Int J Clin Lab Res.27:81-86, 1997). Due to their tissue-specific expression and highexpression levels in cancer, these molecules are currently beinginvestigated as targets for cancer vaccines (Durrant, Anticancer Drugs8:727-733, 1997; Reynolds et al., Int J Cancer 72:972-976, 1997). Theexpression pattern of 24P4C12 provides evidence that it is likewise apotential target for a cancer vaccine approach to prostate cancer andother cancers, as its expression is limited in normal tissues. Itsstructural features as a potential receptor also provides evidence that24P4C12 may be a small molecule target, as well as a target forantibody-based therapeutic strategies.

[0158] Accordingly, therapeutic approaches targeting extracellularportions of 24P4C12, or aimed at inhibiting the activity of the 24P4C12protein are expected to be useful for patients suffering from prostatecancer, testicular cancer, and other cancers expressing 24P4C12. Thetherapeutic approaches aimed at inhibiting the activity of the 24P4C12protein generally fall into two classes. One class comprises variousmethods for inhibiting the binding or association of the 24P4C12 proteinwith its binding partner or with other proteins. Another class comprisesa variety of methods for inhibiting the transcription of the 24P4C12gene or translation of 24P4C12 mRNA.

[0159] 24P4C12 as a Cell Surface Target for Antibody-Based Therapy

[0160] The structural features of 24P4C12 indicate that this molecule islikely a cell surface antigen, providing an attractive target forantibody-based therapeutic strategies. Because 24P4C12 is over-expressedon cancer cells relative to normal cells, systemic administration of24P4C12-immunoreactive compositions would be expected to exhibitrelatively good sensitivity with minimal toxic, non-specific and/ornon-target effects caused by binding of the immunotherapeutic moleculeto non-target organs and tissues. Antibodies specifically reactive withextracellular domains of 24P4C12 can be useful to treat24P4C12-expressing cancers systemically, either as conjugates with atoxin or therapeutic agent, or as naked antibodies capable of inhibitingcell proliferation or function.

[0161] 24P4C12 antibodies can be introduced into a patient such that theantibody binds to 24P4C12 on the cancer cells and mediates thedestruction of the cells and the tumor and/or inhibits the growth of thecells or the tumor. Mechanisms by which such antibodies exert atherapeutic effect may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, modulating the physiologicalfunction of 24P4C12, inhibiting ligand binding or signal transductionpathways, modulating tumor cell differentiation, altering tumorangiogenesis factor profiles, and/or by inducing apoptosis. 24P4C12antibodies can be conjugated to toxic or therapeutic agents and used todeliver the toxic or therapeutic agent directly to 24P4C12-bearing tumorcells. Examples of toxic agents include, but are not limited to,calchemicin, maytansinoids, radioisotopes such as ¹³¹I, ytrium, andbismuth.

[0162] Cancer immunotherapy using anti-24P4C12 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166); Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp., naked antibody;Coulter Pharmaceuticals, Palo Alto, Calif., ¹³¹I-anti-CD20 conjugate),while others involve co-administration of antibodies and othertherapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel(Genentech, Inc.). For treatment of prostate cancer, for example,24P4C12 antibodies can be administered in conjunction with radiation,chemotherapy or hormone ablation.

[0163] Although 24P4C12 antibody therapy may be useful for all stages ofcancer, antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

[0164] It may be desirable for some cancer patients to be evaluated forthe presence and level of 24P4C12 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative 24P4C12imaging, or other techniques capable of reliably indicating the presenceand degree of 24P4C12 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

[0165] Anti-24P4C12 monoclonal antibodies useful in treating prostateand other cancers include those that are capable of initiating a potentimmune response against the tumor and those that are capable of directcytotoxicity. In this regard, anti-24P4C12 monoclonal antibodies (mAbs)may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-24P4C12 mAbs that exert a direct biological effect ontumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-24P4C12 mAb exertsan anti-tumor effect may be evaluated using any number of in vitroassays designed to determine ADCC, ADMMC, complement-mediated celllysis, and so forth, as is generally known in the art.

[0166] The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 24P4C12antigen with high affinity but exhibit low or no antigenicity in thepatient.

[0167] Therapeutic methods of the invention contemplate theadministration of single anti-24P4C12 mAbs as well as combinations, orcocktails, of different mAbs. Such mAb cocktails may have certainadvantages inasmuch as they contain mAbs that target different epitopes,exploit different effector mechanisms or combine directly cytotoxic mAbswith mAbs that rely on immune effector functionality. Such mAbs incombination may exhibit synergistic therapeutic effects. In addition,the administration of anti-24P4C12 mAbs may be combined with othertherapeutic agents, including but not limited to variouschemotherapeutic agents, androgen-blockers, and immune modulators (e.g.,IL-2, GM-CSF). The anti-24P4C12 mAbs may be administered in their“naked” or unconjugated form, or may have therapeutic agents conjugatedto them.

[0168] The anti-24P4C12 antibody formulations may be administered viaany route capable of delivering the antibodies to the tumor site.Potentially effective routes of administration include, but are notlimited to, intravenous, intraperitoneal, intramuscular, intratumor,intradermal, and the like. Treatment will generally involve the repeatedadministration of the anti-24P4C12 antibody preparation via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. Doses in the range of 10-500 mg mAb per week may be effectiveand well tolerated.

[0169] Based on clinical experience with the Herceptin mAb in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV followed by weekly doses ofabout 2 mg/kg IV of the anti-24P4C12 mAb preparation may represent anacceptable dosing regimen. Preferably, the initial loading dose isadministered as a 90 minute or longer infusion. The periodic maintenancedose may be administered as a 30 minute or longer infusion, provided theinitial dose was well tolerated. However, as one of skill in the artwill understand, various factors will influence the ideal dose regimenin a particular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of 24P4C12expression in the patient, the extent of circulating shed 24P4C12antigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

[0170] Optimally, patients should be evaluated for the level ofcirculating shed 24P4C12 antigen in serum in order to assist in thedetermination of the most effective dosing regimen and related factors.Such evaluations may also be used for monitoring purposes throughouttherapy, and may be useful to gauge therapeutic success in combinationwith evaluating other parameters (such as serum PSA levels in prostatecancer therapy).

[0171] Inhibition of 24P4C12 Protein Function

[0172] The invention includes various methods and compositions forinhibiting the binding of 24P4C12 to its binding partner or ligand, orits association with other protein(s) as well as methods for inhibiting24P4C12 function.

[0173] Inhibition of 24P4C12 with Intracellular Antibodies

[0174] In one approach, recombinant vectors encoding single chainantibodies that specifically bind to 24P4C12 may be introduced into24P4C12 expressing cells via gene transfer technologies, wherein theencoded single chain anti-24P4C12 antibody is expressed intracellularly,binds to 24P4C12 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337.

[0175] Single chain antibodies comprise the variable domains of theheavy and light chain joined by a flexible linker polypeptide, and areexpressed as a single polypeptide. Optionally, single chain antibodiesmay be expressed as a single chain variable region fragment joined tothe light chain constant region. Well known intracellular traffickingsignals may be engineered into recombinant polynucleotide vectorsencoding such single chain antibodies in order to precisely target theexpressed intrabody to the desired intracellular compartment. Forexample, intrabodies targeted to the endoplasmic reticulum (ER) may beengineered to incorporate a leader peptide and, optionally, a C-terminalER retention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

[0176] In order to specifically direct the expression of suchintrabodies to particular tumor cells, the transcription of theintrabody may be placed under the regulatory control of an appropriatetumor-specific promoter and/or enhancer. In order to target intrabodyexpression specifically to prostate, for example, the PSA promoterand/or promoter/enhancer may be utilized (See, for example, U.S. Pat.No. 5,919,652).

[0177] Inhibition of 24P4C12 with Recombinant Proteins

[0178] In another approach, recombinant molecules that are capable ofbinding to 24P4C12 thereby preventing 24P4C12 from accessing/binding toits binding partner(s) or associating with other protein(s) are used toinhibit 24P4C12 function. Such recombinant molecules may, for example,contain the reactive part(s) of a 24P4C12 specific antibody molecule. Ina particular embodiment, the 24P4C12 binding domain of a 24P4C12 bindingpartner may be engineered into a dimeric fusion protein comprising two24P4C12 ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion may contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins may be administered in soluble formto patients suffering from a cancer associated with the expression of24P4C12, where the dimeric fusion protein specifically binds to 24P4C12thereby blocking 24P4C12 interaction with a binding partner. Suchdimeric fusion proteins may be further combined into multimeric proteinsusing known antibody linking technologies.

[0179] Inhibition of 24P4C12 Transcription or Translation

[0180] Within another class of therapeutic approaches, the inventionprovides various methods and compositions for inhibiting thetranscription of the 24P4C12 gene. Similarly, the invention alsoprovides methods and compositions for inhibiting the translation of24P4C12 mRNA into protein.

[0181] In one approach, a method of inhibiting the transcription of the24P4C12 gene comprises contacting the 24P4C12 gene with a 24P4C12antisense polynucleotide. In another approach, a method of inhibiting24P4C12 mRNA translation comprises contacting the 24P4C12 mRNA with anantisense polynucleotide. In another approach, a 24P4C12 specificribozyme may be used to cleave the 24P4C12 message, thereby inhibitingtranslation. Such antisense and ribozyme based methods may also bedirected to the regulatory regions of the 24P4C12 gene, such as the24P4C12 promoter and/or enhancer elements. Similarly, proteins capableof inhibiting a 24P4C12 gene transcription factor may be used to inhibit24P4C12 mRNA transcription. The various polynucleotides and compositionsuseful in the aforementioned methods have been described above. The useof antisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

[0182] Other factors that inhibit the transcription of 24P4C12 throughinterfering with 24P4C12 transcriptional activation may also be usefulfor the treatment of cancers expressing 24P4C12. Similarly, factors thatare capable of interfering with 24P4C12 processing may be useful for thetreatment of cancers expressing 24P4C12. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

[0183] General Considerations for Therapeutic Strategies

[0184] Gene transfer and gene therapy technologies may be used fordelivering therapeutic polynucleotide molecules to tumor cellssynthesizing 24P4C12 (i.e., antisense, ribozyme, polynucleotidesencoding intrabodies and other 24P4C12 inhibitory molecules). A numberof gene therapy approaches are known in the art. Recombinant vectorsencoding 24P4C12 antisense polynucleotides, ribozymes, factors capableof interfering with 24P4C12 transcription, and so forth, may bedelivered to target tumor cells using such gene therapy approaches.

[0185] The above therapeutic approaches may be combined with any one ofa wide variety of chemotherapy or radiation therapy regimens. Thesetherapeutic approaches may also enable the use of reduced dosages ofchemotherapy and/or less frequent administration, particularly inpatients that do not tolerate the toxicity of the chemotherapeutic agentwell.

[0186] The anti-tumor activity of a particular composition (e.g.,antisense, ribozyme, intrabody), or a combination of such compositions,may be evaluated using various in vitro and in vivo assay systems. Invitro assays for evaluating therapeutic potential include cell growthassays, soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 24P4C12 to a bindingpartner, etc.

[0187] In vivo, the effect of a 24P4C12 therapeutic composition may beevaluated in a suitable animal model. For example, xenogenic prostatecancer models wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice, are appropriate in relation to prostate cancer andhave been described (Klein et al., 1997, Nature Medicine 3: 402-408).For example, PCT Patent Application WO98/16628, Sawyers et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

[0188] In vivo assays that qualify the promotion of apoptosis may alsobe useful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

[0189] The therapeutic compositions used in the practice of theforegoing methods may be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material that when combined with the therapeuticcomposition retains the anti-tumor function of the therapeuticcomposition and is non-reactive with the patient's immune system.Examples include, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

[0190] Therapeutic formulations may be solubilized and administered viaany route capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile sodium chloride for injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

[0191] Dosages and administration protocols for the treatment of cancersusing the foregoing methods will vary with the method and the targetcancer and will generally depend on a number of other factorsappreciated in the art.

[0192] Cancer Vaccines

[0193] The invention further provides cancer vaccines comprising a24P4C12 protein or fragment thereof, as well as DNA based vaccines.Preferably, the vaccine comprises an immunogenic portion of a 24P4C12protein or polypeptide. In view of the tissue-restricted expression of24P4C12, 24P4C12 cancer vaccines are expected to be effective atspecifically preventing and/or treating 24P4C12 expressing cancerswithout creating non-specific effects on non-target tissues. The use ofa tumor antigen in a vaccine for generating humoral and cell-mediatedimmunity for use in anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int. J. Cancer 63: 231-237; Fong et al.,1997, J. Immunol. 159: 3113-3117). Such methods can be readily practicedby employing a 24P4C12 protein, or fragment thereof, or a24P4C12-encoding nucleic acid molecule and recombinant vectors capableof expressing and appropriately presenting the 24P4C12 immunogen.

[0194] For example, viral gene delivery systems may be used to deliver a24P4C12-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a 24P4C12 protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human 24P4C12 cDNA may be employed. Inanother embodiment, 24P4C12 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a 24P4C12 protein that are capable ofoptimally binding to specified HLA alleles.

[0195] Various ex vivo strategies may also be employed. One approachinvolves the use of dendritic cells to present 24P4C12 antigen to apatient's immune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to present24P4C12 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with24P4C12 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete 24P4C12protein. Yet another embodiment involves engineering the overexpressionof the 24P4C12 gene in dendritic cells using various implementingvectors known in the art, such as adenovirus (Arthur et al., 1997,Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, CancerRes. 56: 3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869), andtumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing 24P4C12 may also be engineered to expressimmune modulators, such as GM-CSF, and used as immunizing agents.

[0196] Anti-idiotypic anti-24P4C12 antibodies can also be used inanti-cancer therapy as a vaccine for inducing an immune response tocells expressing a 24P4C12 protein. Specifically, the generation ofanti-idiotypic antibodies is well known in the art and can readily beadapted to generate anti-idiotypic anti-24P4C12 antibodies that mimic anepitope on a 24P4C12 protein (see, for example, Wagner et al., 1997,Hybridoma 16: 33-40; Foon et al., 1995, J Clin Invest 96: 334-342;Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76). Such ananti-idiotypic antibody can be used in cancer vaccine strategies.

[0197] Genetic immunization methods may be employed to generateprophylactic or therapeutic humoral and cellular immune responsesdirected against cancer cells expressing 24P4C12. Constructs comprisingDNA encoding a 24P4C12 protein/immunogen and appropriate regulatorysequences may be injected directly into muscle or skin of an individual,such that the cells of the muscle or skin take-up the construct andexpress the encoded 24P4C12 protein/immunogen. Expression of the 24P4C12protein immunogen results in the generation of prophylactic ortherapeutic humoral and cellular immunity against prostate cancers.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used (for review, see information and referencespublished at Internet address www.genweb.com).

[0198] Kits

[0199] For use in the diagnostic and therapeutic applications describedor suggested above, kits are also provided by the invention. Such kitsmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a 24P4C12(and/or H38087) protein or a 24P4C12 (and/or H38087) gene or message,respectively. Where the kit utilizes nucleic acid hybridization todetect the target nucleic acid, the kit may also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence and/or a container comprising a reporter-means, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, florescent, or radioisotopelabel.

[0200] The kit of the invention will typically comprise the containerdescribed above and one or more other containers comprising materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use. A label may be present on the on the container toindicate that the composition is used for a specific therapy ornon-therapeutic application, and may also indicate directions for eitherin vivo or in vitro use, such as those described above.

[0201] The 24P4C12 cDNA was deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC; 10801 UniversityBlvd., Manassas, Va. 20110-2209 USA) as plasmids p24P4C12-GTE9 andp24P4C12-GTE5 on Feb. 2 and 26, 1999, respectively, and have beenaccorded ATCC Designation Numbers 207084 and 207129.

EXAMPLES

[0202] Various aspects of the invention are further described andillustrated by way of the several examples that follow, none of whichare intended to limit the scope of the invention.

Example 1

[0203] SSH-Generated Isolation of cDNA Fragment of the 24P4C12 Gene

[0204] Materials and Methods

[0205] LAPC Xenografts

[0206] LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3:402-408; Craftet al., 1999, Cancer Res. 59:5030-5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and AI, respectively) weregrown in intact male SCID mice or in castrated males, respectively, andwere passaged as small tissue chunks in recipient males. LAPC-4 AIxenografts were derived from LAPC-4 AD tumors. To generate the AIxenografts, male mice bearing LAPC AD tumors were castrated andmaintained for 2-3 months. After the LAPC tumors re-grew, the tumorswere harvested and passaged in castrated males or in female SCID mice.

[0207] Cell Lines

[0208] Human cell lines (e.g., HeLa) were obtained from the ATCC andwere maintained in DMEM with 5% fetal calf serum.

[0209] RNA Isolation

[0210] Tumor tissue and cell lines were homogenized in Trizol reagent(Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cellsto isolate total RNA. Poly A RNA was purified from total RNA usingQiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA werequantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzedby gel electrophoresis.

[0211] Oligonucleotides

[0212] The following HPLC purified oligonucleotides were used. DPNCDN(cDNA synthesis primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 34) Adaptor 1:5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NOS: 35, 36,respectively) 3′GGCCCGTCCTAG5′ Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NOS: 37, 38,respectively) 3′CGGCTCCTAG5′ PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′(SEQ ID NO: 39) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ IDNO: 40) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 41)

[0213] Suppression Subtractive Hybridization

[0214] Suppression subtractive hybridization (SSH) was used to identifycDNAs corresponding to 24P4C12s that may be differentially expressed inprostate cancer. The SSH reaction utilized cDNA from two differentprostate tissue sources, subtracting BPH (benign prostatic hyperplasia)cDNA from LAPC-4 AD cDNA. The LAPC-4 AD cDNA was used as the source ofthe “tester”, while the BPH cDNA was used as the source of the “driver”.

[0215] Double stranded cDNAs corresponding to tester and driver cDNAswere synthesized from 2 μg of poly(A)+ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

[0216] Driver cDNA was generated by combining in a 1:1 ratio Dpn IIdigested cDNA from the relevant xenograft source (see above) with a mixof digested cDNAs derived from human benign prostatic hyperplasia (BPH),the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

[0217] Tester cDNA was generated by diluting 1 μl of Dpn II digestedcDNA from the relevant xenograft source (see above) (400 ng) in 5 μl ofwater. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl ofAdaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in atotal volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase(CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heatingat 72° C. for 5 min.

[0218] The first hybridization was performed by adding 1.5 μl (600 ng)of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

[0219] PCR Amplification, Cloning and Sequencing of Gene FragmentsGenerated from SSH

[0220] To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

[0221] The PCR products were inserted into pCR2.1 using the T/A vectorcloning kit (Invitrogen). Transformed E. coli were subjected toblue/white and ampicillin selection. White colonies were picked andarrayed into 96 well plates and were grown in liquid culture overnight.To identify inserts, PCR amplification was performed on 1 ml ofbacterial culture using the conditions of PCR1 and NP1 and NP2 asprimers. PCR products were analyzed using 2% agarose gelelectrophoresis.

[0222] Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

[0223] RT-PCR Expression Analysis

[0224] First strand cDNAs were generated from 1 μg of mRNA with oligo(d)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturers protocol was used and included an incubationfor 50 min at 42° C. with reverse transcriptase followed by RNAse Htreatment at 37° C. for 20 min. After completing the reaction, thevolume was increased to 200 μl with water prior to normalization. Firststrand cDNAs from 16 different normal human tissues were obtained fromClontech.

[0225] Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:42) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 43) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1× PCR buffer(Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1×Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction wasremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: initial denaturation was at 94° C. for15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for2 min. After agarose gel electrophoresis, the band intensities of the283 bp β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization were required to achieveequal band intensities in all tissues after 22 cycles of PCR.

[0226] To determine expression levels of the 24P4C12 gene, 5 μl ofnormalized first strand cDNA was analyzed by PCR using 25, 30, and 35cycles of amplification using the following primer pairs, which weredesigned with the assistance of (MIT; for details, see,www.genome.wi.mit.edu): 5′-agatgaggaggaggacaaaggtg-3′ (SEQ ID NO:44)5′-actgctgggaggagtaccgagtg-3′ (SEQ ID NO:45)

[0227] Semi quantitative expression analysis was achieved by comparingthe PCR products at cycle numbers that give light band intensities.

[0228] Results

[0229] The SSH experiments described in the Materials and Methods,supra, led to the isolation of numerous candidate gene fragment clones(SSH clones). All candidate clones were sequenced and subjected tohomology analysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or northern analysis.

[0230] One of the SHH clones, comprising about 160 bp, encodes aputative open reading frame (ORF) of 53 amino acids and exhibitssignificant homology to an EST derived from a colon tumor library (FIG.1E; SEQ ID NOS: 3, 4). This SSH clone, designated 24P4C12, was used todesign primers for RT-PCR expression analysis of the 24P4C12 gene invarious tissues. RT-PCR analysis showed that 24P4C12 is expressed in allthe LAPC xenografts and normal prostate at approximately equal levels(FIG. 2A). RT-PCR analysis of first strand cDNA derived from 16 normaltissues showed expression in colon, prostate, kidney and lung after 25cycles of amplification (FIGS. 2B and 2C). Northern blot analysis usingthe 24P4C12 SSH fragment as probe shows the highest expression of anapproximately 3 kb 24P4C12 transcript in LAPC-9AD, followed by LAPC-4 AD(FIGS. 3A-3C).

Example 2

[0231] Cloning of Full Length 24P4C12 cDNAs

[0232] Full length cDNAs encoding the 24P4C12 gene were isolated from anormal prostate library and sequenced. Two of the isolated clones,designated 24P4C12-GTE9 (containing most of the coding region of the24P4C12 gene) and 24P4C12-GTE5 (containing the entire coding region ofthe 24P4C12 gene), were deposited as plasmids p24P4C12-GTE9 andp24P4C12-GTE5 with the ATCC (Manassas, Va.) on Feb. 2 and 26, 1999,respectively, and have been accorded ATCC Designation Numbers 207084 and207129, respectively. These two clones, as well as another cloneencoding most of the 24P4C12 coding region, 24P4C12-GTE4, hadoverlapping nucleotide sequences which were combined to generate thecomplete coding and partial non-coding sequence of the 24P4C12 gene asshown in FIGS. 1A-1D (SEQ ID NO: 1).

[0233] The 2587 bp 24P4C12 cDNA sequence shown in FIGS. 1A-1D (SEQ IDNO: 1) encodes an ORF of 710 amino acids with significant homology tothe mouse gene NG22 and the C. elegans gene CEESB82F. An amino acidsequence alignment of the human 24P4C12 protein encoded by the cDNA ofFIGS. 1A-1D (SEQ ID NO: 2) and the murine NG22 gene products is shown inFIGS. 4A-4B.

[0234] NG22 was recently identified as one of many ORFs within a genomicBAC clone that encompasses the MHC class III in the mouse genome. BothNG22 and CEESB82F appear to be genes that contain 12 transmembranedomains. The 24P4C12 cDNA sequence shown in FIGS. 1A-1D (SEQ ID NO: 1)contains 13 potential transmembrane domains. 12-transmembranetransporter proteins are known (Kitty and Amara, 1992, Curr. Opin.Biotechnology 3: 675-682). Due to the putative secondary structure of24P4C12, it is possible that the 24P4C12 protein functions as a cellsurface membrane pump or transporter.

Example 3

[0235] 24P4C12 Gene Expression Analysis

[0236] 24P4C12 mRNA expression in normal human tissues was analyzed bynorthern blotting of multiple tissue blots (Clontech; Palo Alto,Calif.), comprising a total of 16 different normal human tissues, usinglabeled 24P4C12 SSH fragment (Example 1) as a probe. RNA samples werequantitatively normalized with a β-actin probe. Northern blot analysisshowed expression primarily in prostate and colon, with lower expressiondetected in kidney, and significantly lower expression detected inpancreas, lung and placenta.

[0237] To analyze 24P4C12 expression in cancer tissues, northernblotting was performed on RNA derived from the LAPC xenografts, andseveral prostate and non-prostate cancer cell lines. The results showhigh expression levels of 24P4C12 in LAPC-4 AD, LAPC-4 AI, LAPC-9 AD,LNCaP and LAPC-4 cell line (FIG. 3, FIG. 5). Very high levels aredetected in LAPC-3 AI (FIG. 5). Lower levels are detected in LAPC-9 AI.More detailed analysis of the xenografts shows that 24P4C12 is highlyexpressed in the xenografts even when grown within the tibia of mice(FIG. 5). Northern analysis also shows that 24P4C12 is expressed in thenormal prostate and prostate tumor tissues derived from prostate cancerpatients (FIG. 6A). These results suggest that 24P4C12 is a prostategene that is highly expressed in prostate cancer and may have a utilityas a drug or antibody target in prostate cancer.

Example 4

[0238] Generation of 24P4C12 Polyclonal Antibodies

[0239] To generate polyclonal sera to 24P4C12, a peptide was synthesizedcorresponding to amino acids 1-14 (MGGKQRDEDDEAYG; SEQ ID NO: 71) of the24P4C12 protein sequence. This peptide was coupled to Keyhole limpethemacyanin (KLH) and was used to immunize a rabbit as follows. Therabbit was initially immunized with 200 μg of peptide-KLH mixed incomplete Freund's adjuvant. The rabbit was then injected every two weekswith 200 μg of peptide-KLH in incomplete Freund's adjuvant. Bleeds weretaken approximately 7-10 days following each immunization. ELISA andWestern blotting analyses were used to determine titer and specificityof the rabbit serum to the immunizing peptide and 24P4C12 proteinrespectively. Affinity purified anti-24P4C12 polyclonal antibodies wereprepared by passage of crude serum from immunized rabbit over anaffinity matrix comprised of 24P4C12 peptide covalently coupled toAffigel 15 (BioRad). After extensive washing of the matrix with PBS,antibodies specific to 24P4C12 peptide were eluted with low pH glycinebuffer (0.1M, pH 2.5) and dialyzed against PBS.

[0240] Western blot analysis was then performed with anti-24P4C12 pAb of293T cells transiently transfected with 24P4C12 cDNA either in theCMV-driven PCDNA3.1 Myc-His (Invitrogen) or retroviral pSR-alphaexpression vectors. 293T cells were transiently transfected with 10 μgof either empty vector, or with the 24P4C12 cDNA in pCDNA 3.1 CMV-drivenMYC-His (Invitrogen) or pSR-alpha retroviral expression vectors usingthe CaPO4 method. Forty hours following transfection cells wereharvested and lysed in 2× SDS-PAGE sample buffer. Cell lysates in samplebuffer were then subjected to either mild heat denaturation (70° C.) orstrong heat denaturation (100° C.), separated on a 10% SDS-PAGE gel andtransferred to nitrocellulose. Membranes were then subjected to Westernanalysis with 2 μg/ml of an affinity purified rabbit anti-peptide pAbraised to amino acids 1-14 (MGGKQRDEDDEAYG; SEQ ID NO: 71) of 24P4C12.Anti-24P4C12 immunoreactive bands were visualized by incubation withanti-rabbit-HRP conjugated secondary antibody and enhancedchemiluminescence detection.

[0241] Results of the western blot analysis show specific recognition ofa 90 kD band and of a high molecular smear in transfected cells but notin cells transfected with empty vector (FIG. 10A, FIG. 10B). Thecalculated molecular weight of 24P4C12 from the amino acid sequence is79.2 kD. The appearance of a 90 kD band in western analysis of celllysates suggests that 24P4C12 protein contains post-translationalmodifications. Indeed, there are multiple potential N-linkedglycosylation sites predicted from the amino acid sequence. The ratio ofthe high molecular smear is enhanced by high heat (100° C.) denaturationcompared to mild heat (70° C.) denaturation which suggests aggregationof this 12 transmembrane protein upon heat-induced exposure ofhydrophobic sequences. Multiple lower molecular weight bands are alsodetected in cells transfected with the highly expressed pcDNA 3.1 vectorthat may represent degradation products.

Example 5

[0242] Production of Recombinant 24P4C12 in a Mammalian System

[0243] To express recombinant 24P4C12, the full length 24P4C12 cDNA canbe cloned into an expression vector that provides a 6His tag at thecarboxyl-terminus (pCDNA 3.1 myc-his, InVitrogen). The constructs can betransfected into 293T cells. Transfected 293T cell lysates can be probedwith the anti-24P4C12 polyclonal serum described in Example 4 above in aWestern blot.

[0244] The 24P4C12 genes can also be subcloned into the retroviralexpression vector pSRαMSVtkneo and used to establish 24P4C12 expressingcell lines as follows. The 24P4C12 coding sequence (from translationinitiation ATG to the termination codons) is amplified by PCR using dscDNA template from 24P4C12 cDNA. The PCR product is subcloned intopSRαMSVtkneo via the EcoR1(blunt-ended) and Xba 1 restriction sites onthe vector and transformed into DH5α competent cells. Colonies arepicked to screen for clones with unique internal restriction sites onthe cDNA. The positive clone is confirmed by sequencing of the cDNAinsert. Retroviruses may thereafter be used for infection and generationof various cell lines using, for example, NIH 3T3, PC3, and LnCap cells.

Example 6

[0245] Production of Recombinant 24P4C12 in a Baculovirus System

[0246] To generate a recombinant 24P4C12 protein in a baculovirusexpression system, the 24P4C12 cDNA is cloned into the baculovirustransfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag atthe N-terminus Specifically, pBlueBac-24P4C12 is co-transfected withhelper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda)insect cells to generate recombinant baculovirus (see Invitrogeninstruction manual for details). Baculovirus is then collected from cellsupernatant and purified by plaque assay.

[0247] Recombinant 24P4C12 protein is then generated by infection ofHighFive insect cells (4nVitrogen) with the purified baculovirus.Recombinant 24P4C12 protein may be detected using anti-24P4C12 antibody.24P4C12 protein may be purified and used in various cell based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 24P4C12.

Example 7

[0248] Identification & Cloning of H38087, Family Member of 24P4C12

[0249] H38087 was identified as a family member of 24P4C12 by searchingthe dBEST database with the 24P4C12 amino acid sequence using thetblastn tool in NCBI. ESTs that encode protein fragments of homologousproteins were identified. One of these, H38087, was cloned from a testislibrary. The cDNA (clone GTB6) is 2738 bp in size and encodes a 704amino acid protein with 11 putative transmembrane domains (FIGS. 7A-7D;SEQ ID NOS: 6, 7). The 58 base pairs of 5′ untranslated region are veryGC rich (87%), indicating that this gene may contain translationalregulatory elements (FIGS. 7A-7D). The amino acid sequence of 24P4C12(SEQ ID NO: 2) and H38087 (SEQ ID NO: 7) are 44% identical and 56%homologous over the entire sequence (FIG. 8). Expression analysis showsthat H38087 is ubiquitously expressed (FIG. 9). Highest expressionlevels are detected in testis. Expression is also seen in all the LAPCxenografts. Since H38087 is ubiquitously expressed, it could serve as acontrol for testing 24P4C12-specific therapeutics. A 24P4C12-specifictherapeutic that affects H38087 function could be toxic to normal cells.However, a therapeutic that selectively affects 24P4C12, but not H38087,may be less toxic to normal cells. Therefore, H38087 protein is usefulas a pre-clinical testing tool for therapeutic modalities directedtowards 24P4C12.

Example 8

[0250] Identification of Potential Signal Transduction Pathways

[0251] To determine whether 24P4C12 directly or indirectly activatesknown signal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing24P4C12. These transcriptional reporters contain consensus binding sitesfor known transcription factors that lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof their associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

[0252] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

[0253] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

[0254] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

[0255] 4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

[0256] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

[0257] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

[0258] 24P4C12-mediated effects may be assayed in cells showing mRNAexpression. Luciferase reporter plasmids may be introduced by lipidmediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cells extracts with luciferin substrate and luminescenceof the reaction is monitored in a luminometer.

Example 9

[0259] Generation of 24P4C12 Monoclonal Antibodies

[0260] In order to generate 24P4C12 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encompassing a 24P4C12protein is synthesized and used as immunogen. Alternatively, 24P4C12 canbe conveniently expressed in 293T cells transfected with a CMV-drivenexpression vector encoding 24P4C12 with a C-terminal 6× His and MYC tag(pcDNA3.1/mycHIS, Invitrogen). HIS-tagged 24P4C12 expressed in cells canbe purified using a nickel column using standard techniques.

[0261] Balb C mice are initially immunized intraperitoneally with 200 μgof the GST-24P4C12 fusion protein mixed in complete Freund's adjuvant.Mice are subsequently immunized every 2 weeks with 75 μg of GST-24P4C12protein mixed in Freund's incomplete adjuvant for a total of 3immunizations. Reactivity of serum from immunized mice to full length24P4C12 protein is monitored by ELISA using a partially purifiedpreparation of HIS-tagged 24P4C12 protein expressed from 293T cells(Example 5). Mice showing the strongest reactivity are rested for 3weeks and given a final injection of fusion protein in PBS and thensacrificed 4 days later. The spleens of the sacrificed mice are thenharvested and fused to SPO/2 myeloma cells using standard procedures(Harlow and Lane, 1988). Supernatants from growth wells following HATselection are screened by ELISA and Western blot to identify 24P4C12specific antibody producing clones.

[0262] The binding affinity of a 24P4C12 monoclonal antibody may bedetermined using standard technology. Affinity measurements quantify thestrength of antibody to epitope binding and may be used to help definewhich 24P4C12 monoclonal antibodies are preferred for diagnostic ortherapeutic use. The BIAcore system (Uppsala, Sweden) is a preferredmethod for determining binding affinity. The BIAcore system uses surfaceplasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Mortonand Myszka, 1998, Methods in Enzymology 295: 268) to monitorbiomolecular interactions in real time. BIAcore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants.

Example 10

[0263] In Vitro Assays of 24P4C12 Function

[0264] The expression of 24P4C12 in prostate and other cancers providesevidence that this gene has a functional role in tumor progressionand/or tumor initiation. It is possible that 24P4C12 functions as areceptor involved in activating proliferation signals. 24P4C12 functioncan be assessed in mammalian cells using in vitro approaches. Formammalian expression, 24P4C12 can be cloned into a number of appropriatevectors, including pcDNA 3.1 myc-His-tag (Example 5) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using suchexpression vectors, 24P4C12 can be expressed in several cell lines,including PC-3, NIH 3T3, LNCAP and 293T. Expression of 24P4C12 can bemonitored using anti-24P4C12 antibodies and northern blot analysis (seeExamples 4 and 9).

[0265] Mammalian cell lines expressing 24P4C12 can be tested in severalin vitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS;Welch et al., Int. J. Cancer 43: 449457). 24P4C12 cell phenotype iscompared to the phenotype of cells that lack expression of 24P4C12.

[0266] Cell lines expressing 24P4C12 can also be assayed for alterationof invasive and migratory properties by measuring passage of cellsthrough a matrigel coated porous membrane chamber (Becton Dickinson).Passage of cells through the membrane to the opposite side is monitoredusing a fluorescent assay (Becton Dickinson Technical Bulletin #428)using calcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 24P4C12 overexpressing PC3, NIH 3T3 andLNCaP cells. To determine whether 24P4C12-expressing cells havechemoattractant properties, indicator cells are monitored for passagethrough the porous membrane toward a gradient of 24P4C12 conditionedmedia compared to control media. This assay may also be used to qualifyand quantify specific neutralization of the 24P4C12 induced effect bycandidate cancer therapeutic compositions.

[0267] The function of 24P4C12 can be evaluated using anti-sense RNAtechnology coupled to the various functional assays described above,e.g. growth, invasion and migration. Anti-sense RNA oligonucleotides canbe introduced into 24P4C12 expressing cells, thereby preventing theexpression of 24P4C12. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of 24P4C12 expression can be evaluated.

Example 11

[0268] In Vivo Assay for 24P4C12 Tumor Growth Promotion

[0269] The effect of the 24P4C12 protein on tumor cell growth may beevaluated in vivo by gene overexpression in tumor-bearing mice. Forexample, SCID mice can be injected subcutaneously on each flank with1×10⁶ of either PC3, TSUPR1, or DU145 cells containing tkNeo emptyvector or 24P4C12. At least two strategies may be used: (1) Constitutive24P4C12 expression under regulation of a promoter such as a constitutivepromoter obtained from the genomes of viruses such as polyoma virus,fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, provided such promoters arecompatible with the host cell systems, and (2) Regulated expressionunder control of an inducible vector system, such as ecdysone, tet,etc., provided such promoters are compatible with the host cell systems.Tumor volume is then monitored at the appearance of palpable tumors andfollowed over time to determine if 24P4C12 expressing cells grow at afaster rate and whether tumors produced by 24P4C12-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice may be implanted with 1×10⁵ of the same cellsorthotopically to determine if 24P4C12 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow.

[0270] The assay is also useful to determine the 24P4C12 inhibitoryeffect of candidate therapeutic compositions, such as for example,24P4C12 intrabodies, 24P4C12 antisense molecules and ribozymes.

Example 12

[0271] Western Analysis of 24P4C12 Expression in Subcellular Fractions

[0272] Sequence analysis of 24P4C12 revealed the presence of atransmembrane domain. The cellular location of 24P4C12 can be assessedusing subcellular fractionation techniques widely used in cellularbiology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). Prostatecell lines can be separated into nuclear, cytosolic and membranefractions. The expression of 24P4C12 in the different fractions can betested using western blotting techniques.

[0273] Alternatively, to determine the subcellular localization of24P4C12, 293T cells can be transfected with an expression vectorencoding HIS-tagged 24P4C12 (PCDNA 3.1 MYC/HIS, Invitrogen). Thetransfected cells can be harvested and subjected to a differentialsubcellular fractionation protocol as previously described (Pemberton,P. A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.)This protocol separates the cell into fractions enriched for nuclei,heavy membranes (lysosomes, peroxisomes, and mitochondria), lightmembranes (plasma membrane and endoplasmic reticulum), and solubleproteins.

[0274] Throughout this application, various publications are referencedwithin parentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

[0275] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention. These modifications and other embodimentsinclude, but are not limited to, adapting the various methods, assays,molecules and strategies disclosed herein in connection with 24P4C12 foruse with H38087.

1 71 1 2585 DNA Homo sapiens CDS (4)...(2136) 1 gcc atg ggg gga aag cagcgg gac gag gat gac gag gcc tac ggg aag 48 Met Gly Gly Lys Gln Arg AspGlu Asp Asp Glu Ala Tyr Gly Lys 1 5 10 15 cca gtc aaa tac gac ccc tccttt cga ggc ccc atc aag aac aga agc 96 Pro Val Lys Tyr Asp Pro Ser PheArg Gly Pro Ile Lys Asn Arg Ser 20 25 30 tgc aca gat gtc atc tgc tgc gtcctc ttc ctg ctc ttc att cta ggt 144 Cys Thr Asp Val Ile Cys Cys Val LeuPhe Leu Leu Phe Ile Leu Gly 35 40 45 tac atc gtg gtg ggg att gtg gcc tggttg tat gga gac ccc cgg caa 192 Tyr Ile Val Val Gly Ile Val Ala Trp LeuTyr Gly Asp Pro Arg Gln 50 55 60 gtc ctc tac ccc agg aac tct act ggg gcctac tgt ggc atg ggg gag 240 Val Leu Tyr Pro Arg Asn Ser Thr Gly Ala TyrCys Gly Met Gly Glu 65 70 75 aac aaa gat aag ccg tat ctc ctg tac ttc aacatc ttc agc tgc atc 288 Asn Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn IlePhe Ser Cys Ile 80 85 90 95 ctg tcc agc aac atc atc tca gtt gct gag aacggc cta cag tgc ccc 336 Leu Ser Ser Asn Ile Ile Ser Val Ala Glu Asn GlyLeu Gln Cys Pro 100 105 110 aca ccc cag gtg tgt gtg tcc tcc tgc ccg gaggac cca tgg act gtg 384 Thr Pro Gln Val Cys Val Ser Ser Cys Pro Glu AspPro Trp Thr Val 115 120 125 gga aaa aac gag ttc tca cag act gtt ggg gaagtc ttc tat aca aaa 432 Gly Lys Asn Glu Phe Ser Gln Thr Val Gly Glu ValPhe Tyr Thr Lys 130 135 140 aac agg aac ttt tgt ctg cca ggg gta ccc tggaat atg acg gtg atc 480 Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp AsnMet Thr Val Ile 145 150 155 aca agc ctg caa cag gaa ctc tgc ccc agt ttcctc ctc ccc tct gct 528 Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe LeuLeu Pro Ser Ala 160 165 170 175 cca gct ctg ggg cgc tgc ttt cca tgg accaac gtt act cca ccg gcg 576 Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr AsnVal Thr Pro Pro Ala 180 185 190 ctc cca ggg atc acc aat gac acc acc atacag cag ggg atc agc ggt 624 Leu Pro Gly Ile Thr Asn Asp Thr Thr Ile GlnGln Gly Ile Ser Gly 195 200 205 ctt att gac agc ctc aat gcc cga gac atcagt gtt aag atc ttt gaa 672 Leu Ile Asp Ser Leu Asn Ala Arg Asp Ile SerVal Lys Ile Phe Glu 210 215 220 gat ttt gcc cag tcc tgg tat tgg att cttgtt gcc ctg ggg gtg gct 720 Asp Phe Ala Gln Ser Trp Tyr Trp Ile Leu ValAla Leu Gly Val Ala 225 230 235 ctg gtc ttg agc cta ctg ttt atc ttg cttctg cgc ctg gtg gct ggg 768 Leu Val Leu Ser Leu Leu Phe Ile Leu Leu LeuArg Leu Val Ala Gly 240 245 250 255 ccc ctg gtg ctg gtg ctg atc ctg ggagtg ctg ggc gtg ctg gca tac 816 Pro Leu Val Leu Val Leu Ile Leu Gly ValLeu Gly Val Leu Ala Tyr 260 265 270 ggc atc tac tac tgc tgg gag gag taccga gtg ctg cgg gac aag ggc 864 Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr ArgVal Leu Arg Asp Lys Gly 275 280 285 gcc tcc atc tcc cag ctg ggt ttc accacc aac ctc agt gcc tac cag 912 Ala Ser Ile Ser Gln Leu Gly Phe Thr ThrAsn Leu Ser Ala Tyr Gln 290 295 300 agc gtg cag gag acc tgg ctg gcc gccctg atc gtg ttg gcg gtg ctt 960 Ser Val Gln Glu Thr Trp Leu Ala Ala LeuIle Val Leu Ala Val Leu 305 310 315 gaa gcc atc ctg ctg ctg atg ctc atcttc ctg cgg cag cgg att cgt 1008 Glu Ala Ile Leu Leu Leu Met Leu Ile PheLeu Arg Gln Arg Ile Arg 320 325 330 335 att gcc atc gcc ctc ctg aag gaggcc agc aag gct gtg gga cag atg 1056 Ile Ala Ile Ala Leu Leu Lys Glu AlaSer Lys Ala Val Gly Gln Met 340 345 350 atg tct acc atg ttc tac cca ctggtc acc ttt gtc ctc ctc ctc atc 1104 Met Ser Thr Met Phe Tyr Pro Leu ValThr Phe Val Leu Leu Leu Ile 355 360 365 tgc att gcc tac tgg gcc atg actgct ctg tac ctg gct aca tcg ggg 1152 Cys Ile Ala Tyr Trp Ala Met Thr AlaLeu Tyr Leu Ala Thr Ser Gly 370 375 380 caa ccc cag tat gtg ctc tgg gcatcc aac atc agc tcc ccc ggc tgt 1200 Gln Pro Gln Tyr Val Leu Trp Ala SerAsn Ile Ser Ser Pro Gly Cys 385 390 395 gag aaa gtg cca ata aat aca tcatgc aac ccc acg gcc cac ctt gtg 1248 Glu Lys Val Pro Ile Asn Thr Ser CysAsn Pro Thr Ala His Leu Val 400 405 410 415 aac tcc tcg tgc cca ggg ctgatg tgc gtc ttc cag ggc tac tca tcc 1296 Asn Ser Ser Cys Pro Gly Leu MetCys Val Phe Gln Gly Tyr Ser Ser 420 425 430 aaa ggc cta atc caa cgt tctgtc ttc aat ctg caa atc tat ggg gtc 1344 Lys Gly Leu Ile Gln Arg Ser ValPhe Asn Leu Gln Ile Tyr Gly Val 435 440 445 ctg ggg ctc ttc tgg acc cttaac tgg gta ctg gcc ctg ggc caa tgc 1392 Leu Gly Leu Phe Trp Thr Leu AsnTrp Val Leu Ala Leu Gly Gln Cys 450 455 460 gtc ctc gct gga gcc ttt gcctcc ttc tac tgg gcc ttc cac aag ccc 1440 Val Leu Ala Gly Ala Phe Ala SerPhe Tyr Trp Ala Phe His Lys Pro 465 470 475 cag gac atc cct acc ttc ccctta atc tct gcc ttc atc cgc aca ctc 1488 Gln Asp Ile Pro Thr Phe Pro LeuIle Ser Ala Phe Ile Arg Thr Leu 480 485 490 495 cgt tac cac act ggg tcattg gca ttt gga gcc ctc atc ctg acc ctt 1536 Arg Tyr His Thr Gly Ser LeuAla Phe Gly Ala Leu Ile Leu Thr Leu 500 505 510 gtg cag ata gcc cgg gtcatc ttg gag tat att gac cac aag ctc aga 1584 Val Gln Ile Ala Arg Val IleLeu Glu Tyr Ile Asp His Lys Leu Arg 515 520 525 gga gtg cag aac cct gtagcc cgc tgc atc atg tgc tgt ttc aag tgc 1632 Gly Val Gln Asn Pro Val AlaArg Cys Ile Met Cys Cys Phe Lys Cys 530 535 540 tgc ctc tgg tgt ctg gaaaaa ttt atc aag ttc cta aac cgc aat gca 1680 Cys Leu Trp Cys Leu Glu LysPhe Ile Lys Phe Leu Asn Arg Asn Ala 545 550 555 tac atc atg atc gcc atctac ggg aag aat ttc tgt gtc tca gcc aaa 1728 Tyr Ile Met Ile Ala Ile TyrGly Lys Asn Phe Cys Val Ser Ala Lys 560 565 570 575 aat gcg ttc atg ctactc atg cga aac att gtc agg gtg gtc gtc ctg 1776 Asn Ala Phe Met Leu LeuMet Arg Asn Ile Val Arg Val Val Val Leu 580 585 590 gac aaa gtc aca gacctg ctg ctg ttc ttt ggg aag ctg ctg gtg gtc 1824 Asp Lys Val Thr Asp LeuLeu Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605 gga ggc gtg ggg gtcctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1872 Gly Gly Val Gly Val LeuSer Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615 620 ggg ctg ggt aaa gacttt aag agc ccc cac ctc aac tat tac tgg ctg 1920 Gly Leu Gly Lys Asp PheLys Ser Pro His Leu Asn Tyr Tyr Trp Leu 625 630 635 ccc atc atg acc tccatc ctg ggg gcc tat gtc atc gcc agc ggc ttc 1968 Pro Ile Met Thr Ser IleLeu Gly Ala Tyr Val Ile Ala Ser Gly Phe 640 645 650 655 ttc agc gtt ttcggc atg tgt gtg gac acg ctc ttc ctc tgc ttc ctg 2016 Phe Ser Val Phe GlyMet Cys Val Asp Thr Leu Phe Leu Cys Phe Leu 660 665 670 gaa gac ctg gagcgg aac aac ggc tcc ctg gac cgg ccc tac tac atg 2064 Glu Asp Leu Glu ArgAsn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met 675 680 685 tcc aag agc cttcta aag att ctg ggc aag aag aac gag gcg ccc ccg 2112 Ser Lys Ser Leu LeuLys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro 690 695 700 gac aac aag aagagg aag aag tga cagctccggc cctgatccag gactgcaccc 2166 Asp Asn Lys LysArg Lys Lys * 705 710 cacccccacc gtccagccat ccaacctcac ttcgccttacaggtctccat tttgtggtaa 2226 aaaaaggttt taggccaggc gccgtggctc acgcctgtaatccaacactt tgagaggctg 2286 aggcgggcgg atcacctgag tcaggagttc gagaccagcctggccaacat ggtgaaacct 2346 ccgtctctat taaaaataca aaaattagcc gagagtggtggcatgcacct gtcatcccag 2406 ctactcggga ggctgaggca ggagaatcgc ttgaacccgggaggcagagg ttgcagtgag 2466 ccgagatcgc gccactgcac tccaacctgg gtgacagactctgtctccaa aacaaaacaa 2526 acaaacaaaa agattttatt aaagatattt tgttaactcagtaaaaaaaa aaaaaaaaa 2585 2 710 PRT Homo sapiens 2 Met Gly Gly Lys GlnArg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro 1 5 10 15 Val Lys Tyr AspPro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser Cys 20 25 30 Thr Asp Val IleCys Cys Val Leu Phe Leu Leu Phe Ile Leu Gly Tyr 35 40 45 Ile Val Val GlyIle Val Ala Trp Leu Tyr Gly Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro ArgAsn Ser Thr Gly Ala Tyr Cys Gly Met Gly Glu Asn 65 70 75 80 Lys Asp LysPro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser AsnIle Ile Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro GlnVal Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 LysAsn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135 140Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile Thr 145 150155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro Ser Ala Pro165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn Val Thr Pro Pro AlaLeu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly Ile SerGly Leu 195 200 205 Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser Val Lys IlePhe Glu Asp 210 215 220 Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala LeuGly Val Ala Leu 225 230 235 240 Val Leu Ser Leu Leu Phe Ile Leu Leu LeuArg Leu Val Ala Gly Pro 245 250 255 Leu Val Leu Val Leu Ile Leu Gly ValLeu Gly Val Leu Ala Tyr Gly 260 265 270 Ile Tyr Tyr Cys Trp Glu Glu TyrArg Val Leu Arg Asp Lys Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly PheThr Thr Asn Leu Ser Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp LeuAla Ala Leu Ile Val Leu Ala Val Leu Glu 305 310 315 320 Ala Ile Leu LeuLeu Met Leu Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile AlaLeu Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser ThrMet Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 IleAla Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375 380Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys Glu 385 390395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val Asn405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser SerLys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr GlyVal Leu 435 440 445 Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu GlyGln Cys Val 450 455 460 Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala PheHis Lys Pro Gln 465 470 475 480 Asp Ile Pro Thr Phe Pro Leu Ile Ser AlaPhe Ile Arg Thr Leu Arg 485 490 495 Tyr His Thr Gly Ser Leu Ala Phe GlyAla Leu Ile Leu Thr Leu Val 500 505 510 Gln Ile Ala Arg Val Ile Leu GluTyr Ile Asp His Lys Leu Arg Gly 515 520 525 Val Gln Asn Pro Val Ala ArgCys Ile Met Cys Cys Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu LysPhe Ile Lys Phe Leu Asn Arg Asn Ala Tyr 545 550 555 560 Ile Met Ile AlaIle Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe MetLeu Leu Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys ValThr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 GlyVal Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615 620Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu Pro 625 630635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe Phe645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe LeuGlu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr TyrMet Ser 675 680 685 Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu AlaPro Pro Asp 690 695 700 Asn Lys Lys Arg Lys Lys 705 710 3 160 DNA Homosapiens CDS (1)...(160) 3 gat cag ggc ggc cag cca ggt ctc ctg cac gctctg gta ggc act gag 48 Asp Gln Gly Gly Gln Pro Gly Leu Leu His Ala LeuVal Gly Thr Glu 1 5 10 15 gtt ggt ggt gaa acc cag ctg gga gat gga ggcgcc ctc gtc ccg cag 96 Val Gly Gly Glu Thr Gln Leu Gly Asp Gly Gly AlaLeu Val Pro Gln 20 25 30 cac tcg gta ctc ctc cca gca gta gta gat gcc atatgc cag cac gcc 144 His Ser Val Leu Leu Pro Ala Val Val Asp Ala Ile CysGln His Ala 35 40 45 cag cac tcc cag gat c 160 Gln His Ser Gln Asp 50 453 PRT Homo sapiens 4 Asp Gln Gly Gly Gln Pro Gly Leu Leu His Ala LeuVal Gly Thr Glu 1 5 10 15 Val Gly Gly Glu Thr Gln Leu Gly Asp Gly GlyAla Leu Val Pro Gln 20 25 30 His Ser Val Leu Leu Pro Ala Val Val Asp AlaIle Cys Gln His Ala 35 40 45 Gln His Ser Gln Asp 50 5 705 PRT Mouse 5Arg Lys Gln Asn Glu Asn Glu Ala His Gly Asn Ser Ala Lys Tyr Asp 1 5 1015 Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Gly Cys Thr Asp Ile Ile 20 2530 Cys Cys Val Leu Phe Leu Ile Phe Ile Leu Gly Tyr Ile Ile Val Gly 35 4045 Leu Val Ala Trp Val Tyr Gly Asp Pro Arg Gln Val Leu Tyr Pro Arg 50 5560 Asn Ser Thr Gly Ala Tyr Cys Gly Val Gly Asp Asn Lys Asp Lys Pro 65 7075 80 Tyr Val Leu Tyr Phe Asp Ile Leu Ser Cys Ala Ala Ala Ile Asn Ile 8590 95 Ile Ser Ile Ala Glu Asn Gly Leu Gln Cys Pro Thr Pro Gln Val Cys100 105 110 Val Ser Ser Cys Pro Leu Ala Pro Trp Ala Val Glu Val Phe GlnPhe 115 120 125 Ser Lys Thr Val Gly Glu Val Tyr Gly Glu Arg Arg Asn PheCys Leu 130 135 140 Pro Ala Val Ser Pro Asp Met Ile Val Glu Glu Ser LeuGln Lys Gly 145 150 155 160 Leu Cys Pro Arg Phe Leu Leu Pro Ser Thr ProAla Leu Gly Arg Cys 165 170 175 Phe Pro Leu Pro Asn Ile Asn Phe Thr LeuPro Glu Asp Leu Arg Ile 180 185 190 Asn Asn Thr Thr Val Ser Asn Gly IleSer Gly Leu Leu Asp Ser Ile 195 200 205 Asn Ala Arg Asp Val Ser Val LysIle Phe Glu Asp Phe Ala Gln Ser 210 215 220 Trp Tyr Trp Ile Leu Val AlaLeu Gly Val Ala Leu Ala Leu Ser Leu 225 230 235 240 Leu Phe Ile Leu LeuLeu Arg Leu Val Ala Ala Pro Leu Val Leu Leu 245 250 255 Leu Ile Val GlyVal Leu Ala Val Leu Ala Tyr Gly Ile Tyr His Cys 260 265 270 Trp Gln GlnTyr Gln Val Phe Arg Asp Lys Gly Ala Ser Ile Thr Gln 275 280 285 Leu GlyPhe Thr Thr Asn Phe Ser Ala Tyr Gln Ser Val Lys Glu Thr 290 295 300 TrpLeu Ala Ala Leu Ile Val Leu Ala Val Leu Glu Gly Ile Leu Leu 305 310 315320 Leu Met Leu Ile Phe Leu Arg Gln Arg Ile Arg Ile Ala Ile Ala Leu 325330 335 Leu Lys Glu Ala Ser Arg Ala Val Gly Gln Met Met Ser Thr Met Phe340 345 350 Tyr Pro Leu Val Thr Phe Val Leu Leu Val Ile Cys Ile Gly TyrTrp 355 360 365 Ala Val Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln Pro GlnTyr Ile 370 375 380 Tyr Trp Ala Ser Asn Thr Ser Thr Pro Gly Cys Glu AsnVal Pro Val 385 390 395 400 Asn Met Thr Cys Asp Pro Met Ala Pro Leu AsnSer Ser Cys Pro Asn 405 410 415 Leu Lys Cys Val Phe Lys Gly Tyr Ser ThrThr Gly Leu Ala Gln Arg 420 425 430 Ser Leu Phe Asn Leu Gln Ile Tyr GlyVal Leu Gly Leu Phe Trp Thr 435 440 445 Val Asn Trp Val Leu Ala Leu GlyGln Cys Val Leu Ala Gly Ala Phe 450 455 460 Ala Ser Phe Tyr Trp Ala PheHis Lys Pro Arg Asp Ile Pro Thr Phe 465 470 475 480 Pro Leu Ser Ser AlaPhe Ile Arg Thr Leu Arg Tyr His Thr Gly Ser 485 490 495 Leu Ala Phe GlyAla Leu Ile Leu Ser Leu Val Gln Ile Ala Arg Val 500 505 510 Ile Leu GluTyr Ile Asp His Lys Leu Arg Gly Ser Gln Asn Pro Val 515 520 525 Ala ArgCys Ile Ile Cys Cys Phe Lys Cys Cys Leu Trp Cys Leu Glu 530 535 540 LysPhe Ile Lys Phe Leu Asn Arg Asn Ala Tyr Ile Met Ile Ala Ile 545 550 555560 Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys Asn Ala Phe Met Leu Leu 565570 575 Met Arg Asn Val Leu Arg Val Val Val Leu Asp Lys Val Thr Asp Leu580 585 590 Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly Gly Val Gly ValLeu 595 600 605 Ser Phe Phe Phe Phe Ser Gly Arg Ile Lys Gly Leu Gly LysAsp Phe 610 615 620 Glu Asn Pro Asn Leu Asn Tyr Tyr Trp Leu Pro Ile MetThr Ser Ile 625 630 635 640 Met Gly Ala Tyr Val Ile Ala Ser Gly Phe PheSer Val Phe Gly Met 645 650 655 Cys Val Asp Thr Leu Phe Leu Cys Phe LeuGlu Asp Leu Glu Arg Asn 660 665 670 Asp Gly Ser Gln Glu Arg Pro Tyr TyrMet Pro Lys Ala Leu Leu Lys 675 680 685 Ile Leu Gly Lys Lys Asn Glu AlaPro Thr Gly Gly Lys Thr Arg Lys 690 695 700 Lys 705 6 2737 DNA Homosapiens CDS (58)...(2172) 6 gcccgcccgg gctggggtcg cgctggctcg gactccgctccccgccccgc cgcggcc atg 60 Met 1 gag gac gag cgg aaa aac gga gcc tac ggaacg cca cag aag tat gat 108 Glu Asp Glu Arg Lys Asn Gly Ala Tyr Gly ThrPro Gln Lys Tyr Asp 5 10 15 ccc act ttc aaa gga ccc att tac aat agg ggctgc acg gat atc ata 156 Pro Thr Phe Lys Gly Pro Ile Tyr Asn Arg Gly CysThr Asp Ile Ile 20 25 30 tgc tgt gtg ttc ctg ctc ctg gcc att gtg ggc tacgtg gct gta ggc 204 Cys Cys Val Phe Leu Leu Leu Ala Ile Val Gly Tyr ValAla Val Gly 35 40 45 atc ata gcc tgg act cat gga gac cct cga aag gtg atctac ccc act 252 Ile Ile Ala Trp Thr His Gly Asp Pro Arg Lys Val Ile TyrPro Thr 50 55 60 65 gat agc cgg ggc gag ttc tgc ggg cag aag ggc aca aaaaac gag aac 300 Asp Ser Arg Gly Glu Phe Cys Gly Gln Lys Gly Thr Lys AsnGlu Asn 70 75 80 aaa ccc tat ctg ttt tat ttc aac att gtg aaa tgt gcc agcccc ctg 348 Lys Pro Tyr Leu Phe Tyr Phe Asn Ile Val Lys Cys Ala Ser ProLeu 85 90 95 gtt ctg ctg gaa ttc caa tgt ccc act ccc cag atc tgc gtg gaaaaa 396 Val Leu Leu Glu Phe Gln Cys Pro Thr Pro Gln Ile Cys Val Glu Lys100 105 110 tgc ccc gac cgc tac ctc acg tac ctg aat gct cgc agc tcc cgggac 444 Cys Pro Asp Arg Tyr Leu Thr Tyr Leu Asn Ala Arg Ser Ser Arg Asp115 120 125 ttt gag tac tat aag cag ttc tgt gtt cct ggc ttc aag aac aataaa 492 Phe Glu Tyr Tyr Lys Gln Phe Cys Val Pro Gly Phe Lys Asn Asn Lys130 135 140 145 gga gtg gct gag gtg ctt cga gat ggt gac tgc cct gct gtcctc atc 540 Gly Val Ala Glu Val Leu Arg Asp Gly Asp Cys Pro Ala Val LeuIle 150 155 160 ccc agc aaa ccc ttg gcc cgg aga tgc ttc ccc gct atc cacgcc tac 588 Pro Ser Lys Pro Leu Ala Arg Arg Cys Phe Pro Ala Ile His AlaTyr 165 170 175 aag ggt gtc ctg atg gtg ggc aat gag acg acc tat gag gatggg cat 636 Lys Gly Val Leu Met Val Gly Asn Glu Thr Thr Tyr Glu Asp GlyHis 180 185 190 ggc tcc cgg aaa aac atc aca gac ctg gtg gag ggc gcc aagaaa gcc 684 Gly Ser Arg Lys Asn Ile Thr Asp Leu Val Glu Gly Ala Lys LysAla 195 200 205 aat gga gtc cta gag gcg cgg caa ctc gcc atg cgc ata tttgaa gat 732 Asn Gly Val Leu Glu Ala Arg Gln Leu Ala Met Arg Ile Phe GluAsp 210 215 220 225 tac acc gtc tct tgg tac tgg att atc ata ggc ctg gtcatt gcc atg 780 Tyr Thr Val Ser Trp Tyr Trp Ile Ile Ile Gly Leu Val IleAla Met 230 235 240 gcg atg agc ctc ctg ttc atc atc ctg ctt cgc ttc ctggct ggt att 828 Ala Met Ser Leu Leu Phe Ile Ile Leu Leu Arg Phe Leu AlaGly Ile 245 250 255 atg gtc tgg gtg atg atc atc atg gtg att ctg gtg ctgggc tac gga 876 Met Val Trp Val Met Ile Ile Met Val Ile Leu Val Leu GlyTyr Gly 260 265 270 ata ttt cac tgc tac atg gag tac tcc cga ctg cgt ggtgag gcc ggc 924 Ile Phe His Cys Tyr Met Glu Tyr Ser Arg Leu Arg Gly GluAla Gly 275 280 285 tct gat gtc tct ttg gtg gac ctc ggc ttt cag acg gatttc cgg gtg 972 Ser Asp Val Ser Leu Val Asp Leu Gly Phe Gln Thr Asp PheArg Val 290 295 300 305 tac ctg cac tta cgg cag acc tgg ttg gcc ttt atgatc att ctg agt 1020 Tyr Leu His Leu Arg Gln Thr Trp Leu Ala Phe Met IleIle Leu Ser 310 315 320 atc ctt gaa gtc att atc atc ttg ctg ctc atc tttctc cgg aag aga 1068 Ile Leu Glu Val Ile Ile Ile Leu Leu Leu Ile Phe LeuArg Lys Arg 325 330 335 att ctc atc gcg att gca ctc atc aaa gaa gcc agcagg gct gtg gga 1116 Ile Leu Ile Ala Ile Ala Leu Ile Lys Glu Ala Ser ArgAla Val Gly 340 345 350 tac gtc atg tgc tcc ttg ctc tac cca ctg gtc accttc ttc ttg ctg 1164 Tyr Val Met Cys Ser Leu Leu Tyr Pro Leu Val Thr PhePhe Leu Leu 355 360 365 tgc ctc tgc atc gcc tac tgg gcc agc act gct gtcttc ctg tcc act 1212 Cys Leu Cys Ile Ala Tyr Trp Ala Ser Thr Ala Val PheLeu Ser Thr 370 375 380 385 tcc aac gaa gcg gtc tat aag atc ttt gat gacagc ccc tgc cca ttt 1260 Ser Asn Glu Ala Val Tyr Lys Ile Phe Asp Asp SerPro Cys Pro Phe 390 395 400 act gcg aaa acc tgc aac cca gag acc ttc ccctcc tcc cat gag tcc 1308 Thr Ala Lys Thr Cys Asn Pro Glu Thr Phe Pro SerSer His Glu Ser 405 410 415 cgc caa tgc ccc aat gcc cgt tgc cag ttc gtcttc tac ggt ggt gag 1356 Arg Gln Cys Pro Asn Ala Arg Cys Gln Phe Val PheTyr Gly Gly Glu 420 425 430 tcg ggc tac cac cgg gcc ctg ctg ggc ctg cagatc ttc aat gcc ttc 1404 Ser Gly Tyr His Arg Ala Leu Leu Gly Leu Gln IlePhe Asn Ala Phe 435 440 445 atg ttc ttc tgg ttg gcc aac ttc gtg ctg gcgctg ggc cag gtc acg 1452 Met Phe Phe Trp Leu Ala Asn Phe Val Leu Ala LeuGly Gln Val Thr 450 455 460 465 ctg gcc ggg gcc ttt gcc tcc tac tac tgggcc ctg cgc aag ccg gac 1500 Leu Ala Gly Ala Phe Ala Ser Tyr Tyr Trp AlaLeu Arg Lys Pro Asp 470 475 480 gac ctg ccg gcc ttc ccg ctc ttc tct gccttt ggc cgg gcg ctc agg 1548 Asp Leu Pro Ala Phe Pro Leu Phe Ser Ala PheGly Arg Ala Leu Arg 485 490 495 tac cac aca ggc tcc ctg gcc ttt ggc gcgctc atc ctg gcc att gtg 1596 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala LeuIle Leu Ala Ile Val 500 505 510 cag atc atc cgt gtg ata ctc gag tac ctggat cag cgg ctg aaa gct 1644 Gln Ile Ile Arg Val Ile Leu Glu Tyr Leu AspGln Arg Leu Lys Ala 515 520 525 gca gag aac aag ttt gcc aag tgc ctc atgacc tgt ctc aaa tgc tgc 1692 Ala Glu Asn Lys Phe Ala Lys Cys Leu Met ThrCys Leu Lys Cys Cys 530 535 540 545 ttc tgg tgc ctg gag aag ttc atc aaattc ctt aat agg aat gcc tac 1740 Phe Trp Cys Leu Glu Lys Phe Ile Lys PheLeu Asn Arg Asn Ala Tyr 550 555 560 atc atg att gcc atc tac ggc acc aatttc tgc acc tcg gcc agg aat 1788 Ile Met Ile Ala Ile Tyr Gly Thr Asn PheCys Thr Ser Ala Arg Asn 565 570 575 gcc ttc ttc ctg ctc atg aga aac atcatc aga gtg gct gtc ctg gat 1836 Ala Phe Phe Leu Leu Met Arg Asn Ile IleArg Val Ala Val Leu Asp 580 585 590 aaa gtt act gac ttc ctc ttc ctg ttgggc aaa ctt ctg atc gtt ggt 1884 Lys Val Thr Asp Phe Leu Phe Leu Leu GlyLys Leu Leu Ile Val Gly 595 600 605 agt gtg ggg atc ctg gct ttc ttc ttcttc acc cac cgt atc agg atc 1932 Ser Val Gly Ile Leu Ala Phe Phe Phe PheThr His Arg Ile Arg Ile 610 615 620 625 gtg cag gat aca gca cca ccc ctcaat tat tac tgg gtt cct ata ctg 1980 Val Gln Asp Thr Ala Pro Pro Leu AsnTyr Tyr Trp Val Pro Ile Leu 630 635 640 acg gtg atc gtt ggc tcc tac ttgatt gca cac ggt ttc ttc agc gtc 2028 Thr Val Ile Val Gly Ser Tyr Leu IleAla His Gly Phe Phe Ser Val 645 650 655 tat ggc atg tgt gtg gac acg ctgttc ctc tgc ttc ttg gag gac ctg 2076 Tyr Gly Met Cys Val Asp Thr Leu PheLeu Cys Phe Leu Glu Asp Leu 660 665 670 gag agg aat gac ggc tcg gcc gagagg cct tac ttc atg tct tcc acc 2124 Glu Arg Asn Asp Gly Ser Ala Glu ArgPro Tyr Phe Met Ser Ser Thr 675 680 685 ctc aag aaa ctc ttg aac aag accaac aag aag gca gcg gag tcc tga 2172 Leu Lys Lys Leu Leu Asn Lys Thr AsnLys Lys Ala Ala Glu Ser * 690 695 700 aggccccgtg ctccccacct ctcaaggagtctcatgccgc agggtgctca gtagctgggt 2232 ctgttccccc agccccttgg gctcacctgaagtcctatca ctgccgctct gcccctcccc 2292 atgagccaga tcccaccagt ttctggacgtggagagtctg gggcatctcc ttcttatgcc 2352 aaggggcgct tggagttttc atggctgcccctccagactg cgagaaacaa gtaaaaaccc 2412 attggggcct cttgatgtct gggatggcacgtggcccgac ctccacaagc tccctcatgc 2472 ttcctgtccc ccgcttacac gacaacgggccagaccacgg gaaggacggt gtttgtgtct 2532 gagggagctg ctggccacag tgaacacccacgtttattcc tgcctgctcc ggccaggact 2592 gaaccccttc tccacacctg aacagttggctcaagggcca ccagaagcat ttctttatta 2652 ttattatttt ttaacctgga catgcattaaagggtctatt agctttcaaa aaaaaaaaaa 2712 aaaaaaaaaa aaaaaaaaaa aaaaa 2737 7704 PRT Homo sapiens 7 Met Glu Asp Glu Arg Lys Asn Gly Ala Tyr Gly ThrPro Gln Lys Tyr 1 5 10 15 Asp Pro Thr Phe Lys Gly Pro Ile Tyr Asn ArgGly Cys Thr Asp Ile 20 25 30 Ile Cys Cys Val Phe Leu Leu Leu Ala Ile ValGly Tyr Val Ala Val 35 40 45 Gly Ile Ile Ala Trp Thr His Gly Asp Pro ArgLys Val Ile Tyr Pro 50 55 60 Thr Asp Ser Arg Gly Glu Phe Cys Gly Gln LysGly Thr Lys Asn Glu 65 70 75 80 Asn Lys Pro Tyr Leu Phe Tyr Phe Asn IleVal Lys Cys Ala Ser Pro 85 90 95 Leu Val Leu Leu Glu Phe Gln Cys Pro ThrPro Gln Ile Cys Val Glu 100 105 110 Lys Cys Pro Asp Arg Tyr Leu Thr TyrLeu Asn Ala Arg Ser Ser Arg 115 120 125 Asp Phe Glu Tyr Tyr Lys Gln PheCys Val Pro Gly Phe Lys Asn Asn 130 135 140 Lys Gly Val Ala Glu Val LeuArg Asp Gly Asp Cys Pro Ala Val Leu 145 150 155 160 Ile Pro Ser Lys ProLeu Ala Arg Arg Cys Phe Pro Ala Ile His Ala 165 170 175 Tyr Lys Gly ValLeu Met Val Gly Asn Glu Thr Thr Tyr Glu Asp Gly 180 185 190 His Gly SerArg Lys Asn Ile Thr Asp Leu Val Glu Gly Ala Lys Lys 195 200 205 Ala AsnGly Val Leu Glu Ala Arg Gln Leu Ala Met Arg Ile Phe Glu 210 215 220 AspTyr Thr Val Ser Trp Tyr Trp Ile Ile Ile Gly Leu Val Ile Ala 225 230 235240 Met Ala Met Ser Leu Leu Phe Ile Ile Leu Leu Arg Phe Leu Ala Gly 245250 255 Ile Met Val Trp Val Met Ile Ile Met Val Ile Leu Val Leu Gly Tyr260 265 270 Gly Ile Phe His Cys Tyr Met Glu Tyr Ser Arg Leu Arg Gly GluAla 275 280 285 Gly Ser Asp Val Ser Leu Val Asp Leu Gly Phe Gln Thr AspPhe Arg 290 295 300 Val Tyr Leu His Leu Arg Gln Thr Trp Leu Ala Phe MetIle Ile Leu 305 310 315 320 Ser Ile Leu Glu Val Ile Ile Ile Leu Leu LeuIle Phe Leu Arg Lys 325 330 335 Arg Ile Leu Ile Ala Ile Ala Leu Ile LysGlu Ala Ser Arg Ala Val 340 345 350 Gly Tyr Val Met Cys Ser Leu Leu TyrPro Leu Val Thr Phe Phe Leu 355 360 365 Leu Cys Leu Cys Ile Ala Tyr TrpAla Ser Thr Ala Val Phe Leu Ser 370 375 380 Thr Ser Asn Glu Ala Val TyrLys Ile Phe Asp Asp Ser Pro Cys Pro 385 390 395 400 Phe Thr Ala Lys ThrCys Asn Pro Glu Thr Phe Pro Ser Ser His Glu 405 410 415 Ser Arg Gln CysPro Asn Ala Arg Cys Gln Phe Val Phe Tyr Gly Gly 420 425 430 Glu Ser GlyTyr His Arg Ala Leu Leu Gly Leu Gln Ile Phe Asn Ala 435 440 445 Phe MetPhe Phe Trp Leu Ala Asn Phe Val Leu Ala Leu Gly Gln Val 450 455 460 ThrLeu Ala Gly Ala Phe Ala Ser Tyr Tyr Trp Ala Leu Arg Lys Pro 465 470 475480 Asp Asp Leu Pro Ala Phe Pro Leu Phe Ser Ala Phe Gly Arg Ala Leu 485490 495 Arg Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Ala Ile500 505 510 Val Gln Ile Ile Arg Val Ile Leu Glu Tyr Leu Asp Gln Arg LeuLys 515 520 525 Ala Ala Glu Asn Lys Phe Ala Lys Cys Leu Met Thr Cys LeuLys Cys 530 535 540 Cys Phe Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu AsnArg Asn Ala 545 550 555 560 Tyr Ile Met Ile Ala Ile Tyr Gly Thr Asn PheCys Thr Ser Ala Arg 565 570 575 Asn Ala Phe Phe Leu Leu Met Arg Asn IleIle Arg Val Ala Val Leu 580 585 590 Asp Lys Val Thr Asp Phe Leu Phe LeuLeu Gly Lys Leu Leu Ile Val 595 600 605 Gly Ser Val Gly Ile Leu Ala PhePhe Phe Phe Thr His Arg Ile Arg 610 615 620 Ile Val Gln Asp Thr Ala ProPro Leu Asn Tyr Tyr Trp Val Pro Ile 625 630 635 640 Leu Thr Val Ile ValGly Ser Tyr Leu Ile Ala His Gly Phe Phe Ser 645 650 655 Val Tyr Gly MetCys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu Asp 660 665 670 Leu Glu ArgAsn Asp Gly Ser Ala Glu Arg Pro Tyr Phe Met Ser Ser 675 680 685 Thr LeuLys Lys Leu Leu Asn Lys Thr Asn Lys Lys Ala Ala Glu Ser 690 695 700 8 4PRT Homo sapiens 8 Asn Arg Ser Cys 1 9 4 PRT Homo sapiens 9 Asn Ser ThrGly 1 10 4 PRT Homo sapiens 10 Asn Met Thr Val 1 11 4 PRT Homo sapiens11 Asn Asp Thr Thr 1 12 4 PRT Homo sapiens 12 Asn Leu Ser Ala 1 13 4 PRTHomo sapiens 13 Asn Ile Ser Ser 1 14 4 PRT Homo sapiens 14 Asn Thr SerCys 1 15 4 PRT Homo sapiens 15 Asn Ser Ser Cys 1 16 4 PRT Homo sapiens16 Asn Gly Ser Leu 1 17 4 PRT Homo sapiens 17 Ser Cys Thr Asp 1 18 4 PRTHomo sapiens 18 Ser Val Ala Glu 1 19 4 PRT Homo sapiens 19 Ser Cys ProGlu 1 20 4 PRT Homo sapiens 20 Thr Val Gly Glu 1 21 4 PRT Homo sapiens21 Ser Val Gln Glu 1 22 8 PRT Homo sapiens 22 Arg Asp Glu Asp Asp GluAla Tyr 1 5 23 6 PRT Homo sapiens 23 Gly Ala Tyr Cys Gly Met 1 5 24 6PRT Homo sapiens 24 Gly Met Gly Glu Asn Lys 1 5 25 6 PRT Homo sapiens 25Gly Val Pro Trp Asn Met 1 5 26 6 PRT Homo sapiens 26 Gly Leu Ile Asp SerLeu 1 5 27 6 PRT Homo sapiens 27 Gly Ile Tyr Tyr Cys Trp 1 5 28 6 PRTHomo sapiens 28 Gly Ala Ser Ile Ser Gln 1 5 29 6 PRT Homo sapiens 29 GlyGln Met Met Ser Thr 1 5 30 6 PRT Homo sapiens 30 Gly Leu Phe Trp Thr Leu1 5 31 6 PRT Homo sapiens 31 Gly Ala Phe Ala Ser Phe 1 5 32 4 PRT Homosapiens 32 Leu Gly Lys Lys 1 33 21 PRT Homo sapiens 33 Leu Phe Ile LeuLeu Leu Arg Leu Val Ala Gly Pro Leu Val Leu Val 1 5 10 15 Ile Leu GlyVal Leu 20 34 14 DNA Artificial sequence cDNA synthesis primer 34ttttgatcaa gctt 14 35 42 DNA Artificial sequence Adaptor 1 35 ctaatacgactcactatagg gctcgagcgg ccgcccgggc ag 42 36 12 DNA Artificial sequenceAdaptor 1 36 gatcctgccc gg 12 37 40 DNA Artificial sequence Adaptor 2 37gtaatacgac tcactatagg gcagcgtggt cgcggccgag 40 38 10 DNA Artificialsequence Adaptor 2 38 gatcctcggc 10 39 22 DNA Artificial sequence PCRprimer 1 39 ctaatacgac tcactatagg gc 22 40 22 DNA Artificial sequenceNested primer 1 40 tcgagcggcc gcccgggcag ga 22 41 20 DNA Artificialsequence Nested primer 2 41 agcgtggtcg cggccgagga 20 42 25 DNAArtificial sequence Primer 42 atatcgccgc gctcgtcgtc gacaa 25 43 26 DNAArtificial sequence Primer 43 agccacacgc agctcattgt agaagg 26 44 23 DNAArtificial sequence Primer 44 agatgaggag gaggacaaag gtg 23 45 23 DNAArtificial sequence Primer 45 actgctggga ggagtaccga gtg 23 46 4 PRT Homosapiens 46 Asn Glu Thr Thr 1 47 4 PRT Homo sapiens 47 Asn Ile Thr Asp 148 4 PRT Homo sapiens 48 Asn Lys Thr Asn 1 49 4 PRT Homo sapiens 49 ThrHis Gly Asp 1 50 4 PRT Homo sapiens 50 Ser Arg Gly Glu 1 51 4 PRT Homosapiens 51 Thr Lys Asn Glu 1 52 4 PRT Homo sapiens 52 Ser Ser Arg Asp 153 4 PRT Homo sapiens 53 Thr Thr Tyr Glu 1 54 4 PRT Homo sapiens 54 ThrTyr Glu Asp 1 55 4 PRT Homo sapiens 55 Ser Leu Val Asp 1 56 4 PRT Homosapiens 56 Ser Ile Leu Glu 1 57 4 PRT Homo sapiens 57 Thr Ser Asn Glu 158 4 PRT Homo sapiens 58 Ser Ser His Glu 1 59 9 PRT Homo sapiens 59 ArgSer Ser Arg Asp Phe Glu Tyr Tyr 1 5 60 6 PRT Homo sapiens 60 Gly Gln LysGly Thr Lys 1 5 61 6 PRT Homo sapiens 61 Gly Asn Glu Thr Thr Tyr 1 5 626 PRT Homo sapiens 62 Gly Ser Arg Lys Asn Ile 1 5 63 6 PRT Homo sapiens63 Gly Ala Lys Lys Ala Asn 1 5 64 6 PRT Homo sapiens 64 Gly Val Leu GluAla Arg 1 5 65 6 PRT Homo sapiens 65 Gly Leu Val Ile Ala Met 1 5 66 6PRT Homo sapiens 66 Gly Ile Phe His Cys Tyr 1 5 67 6 PRT Homo sapiens 67Gly Ser Asp Val Ser Leu 1 5 68 6 PRT Homo sapiens 68 Gly Gly Glu Ser GlyTyr 1 5 69 6 PRT Homo sapiens 69 Gly Ala Phe Ala Ser Tyr 1 5 70 6 PRTHomo sapiens 70 Gly Thr Asn Phe Cys Thr 1 5 71 14 PRT Homo sapiens 71Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly 1 5 10

1. A polynucleotide that encodes a 24P4C12 polypeptide, wherein thepolynucleotide is selected from the group consisting of: (a) apolynucleotide having the sequence as shown in FIGS. 1A-1D (SEQ IDNO: 1) or FIG. 1E (SEQ ID NO: 3), wherein T can also be U; (b) apolynucleotide having the sequence as shown in FIGS. 1A-1D (SEQ ID NO:1), from nucleotide residue number 6 through nucleotide residue number2138, wherein T can also be U; (c) a polynucleotide encoding a 24P4C12polypeptide whose sequence is encoded by a cDNA contained in theplasmids designated p24P4C12-GTE5 or p24P4C12-GTE9 deposited withAmerican Type Culture Collection as Designation Nos. 207129 and 207084,respectively; (d) a polynucleotide encoding a 24P4C12 protein having theamino acid sequence shown in FIGS. 1A-1D (SEQ ID NO: 2) or FIG. 1E (SEQID NO: 4); and (e) a polynucleotide that is fully complementary to apolynucleotide of any one of (a)-(d).
 2. A polynucleotide that encodes apolypeptide that is at least 90% identical to the amino acid sequenceshown in FIGS. 1A-1D (SEQ ID NO: 2) or FIG. 1E (SEQ ID NO: 4) over itsentire length.
 3. A polynucleotide that encodes a 24P4C12 polypeptide,wherein the polypeptide includes an amino acid sequence selected fromthe group consisting of NRSC (SEQ ID NO: 8), NSTG (SEQ ID NO: 9), NMTV(SEQ ID NO: 10), NDTT (SEQ ID NO: 11), NLSA (SEQ ID NO: 12), NISS (SEQID NO: 13), NTSC (SEQ ID NO: 14), NSSC (SEQ ID NO: 15), NGSL (SEQ ID NO:16), SFR, SVK, SSK, TLR, SAK, SGR, SCTD (SEQ ID NO: 17), SVAE (SEQ IDNO: 18), SCPE (SEQ ID NO: 19), TVGE (SEQ ID NO: 20), SVQE (SEQ ID NO:21), RDEDDEAY (SEQ ID NO: 22), GAYCGM (SEQ ID NO: 23), GMGENK (SEQ IDNO: 24), GVPWNM (SEQ ID NO: 25), GLIDSL (SEQ ID NO: 26), GIYYCW (SEQ IDNO: 27), GASISQ (SEQ ID NO: 28), GQMMST (SEQ ID NO: 29), GLFWTL (SEQ IDNO: 30), GAFASF (SEQ ID NO: 31), LGKK (SEQ ID NO: 32), andLFILLLRLVAGPLVLVILGVL (SEQ ID NO: 33).
 4. A polynucleotide that encodesa 24P4C12 polypeptide, wherein the polypeptide includes a transmembranedomain shown in FIGS. 1A-1D.
 5. A polynucleotide of any one of claims1-4 that is labeled with a detectable marker.
 6. A recombinantexpression vector that contains a polynucleotide of any one of claims1-4.
 7. A host cell that contains an expression vector of claim
 6. 8. Aprocess for producing a 24P4C12 polypeptide comprising culturing a hostcell of claim 7 under conditions sufficient for the production of thepolypeptide.
 9. The process of claim 8, further comprising recoveringthe 24P4C12 polypeptide so produced.
 10. A 24P4C12 polypeptide producedby the process of claim
 8. 11. A 24P4C12 polypeptide encoded by thepolynucleotide of any one of claims 1-4.
 12. A polypeptide comprising atleast 15 contiguous amino acids of the polypeptide of claim
 11. 13. Anantibody or fragment thereof that specifically binds to the 24P4C12polypeptide of claim
 10. 14. The antibody or fragment thereof of claim13, which is monoclonal.
 15. The antibody or fragment thereof of claim13, which is polyclonal.
 16. A recombinant protein comprising theantigen binding region of a monoclonal antibody of claim
 14. 17. Theantibody or fragment thereof of claim 13, which is labeled with adetectable marker.
 18. The antibody or fragment thereof of claim 17,wherein the detectable marker is selected from the group consisting of aradioisotope, fluorescent compound, bioluminescent compound,chemiluminescent compound, metal chelator or enzyme.
 19. The antibodyfragment of claim 13, which is an Fab, F(ab′)2, Fv or Sfv fragment. 20.The antibody or fragment thereof of claim 13, which is a human antibody.21. The antibody or fragment thereof of claim 13, which is conjugated toa toxin or a therapeutic agent.
 22. The antibody of claim 13, whichcomprises murine antigen binding region residues and human antibodyresidues.
 23. A transgenic animal producing a monoclonal antibody ofclaim
 20. 24. A hybridoma producing a monoclonal antibody of claim 14.25. A single chain monoclonal antibody that comprises the variabledomains of the heavy and light chains of a monoclonal antibody of claim14.
 26. A vector comprising a polynucleotide encoding a single chainmonoclonal antibody of claim
 25. 27. An assay for detecting the presenceof a 24P4C12 protein in a biological sample comprising contacting thesample with an antibody or fragment thereof of claim 17, and detectingthe binding of 24P4C12 protein in the sample thereto.
 28. An assay fordetecting the presence of a 24P4C12 polynucleotide in a biologicalsample, comprising (a) contacting the sample with a polynucleotide probethat specifically hybridizes to the polynucleotide of claim 1; and (b)detecting the presence of a hybridization complex formed by thehybridization of the probe with 24P4C12 polynucleotide in the sample,wherein the presence of the hybridization complex indicates the presenceof 24P4C12 polynucleotide within the sample.
 29. An assay for detectingthe presence of 24P4C12 mRNA in a biological sample comprising: (a)producing cDNA from the sample by reverse transcription using at leastone primer; (b) amplifying the cDNA so produced using 24P4C12polynucleotides as sense and antisense primers to amplify 24P4C12 cDNAstherein; (c) detecting the presence of the amplified 24P4C12 cDNA,wherein the 24P4C12 polynucleotides used as the sense and antisenseprobes are capable of amplifying the 24P4C12 cDNA contained within aplasmids designated p24P4C12-GTE5 or p24P4C12-GTE9, deposited withAmerican Type Culture Collection as Designation Nos. 207129 and 207084,respectively.
 30. A method of detecting the presence of a cancerexpressing 24P4C12 protein that comprises determining the level of24P4C12 protein expressed by cells in a test tissue sample from anindividual and comparing the level so determined to the level of 24P4C12expressed in a corresponding normal sample, the presence of elevated24P4C12 protein in the test sample relative to the normal sampleproviding an indication of the presence of such cancer in theindividual.
 31. A method of monitoring 24P4C12 gene products comprisingdetermining the status of 24P4C12 gene products expressed by cells in atest tissue sample from an individual and comparing the status sodetermined to the status of 24P4C12 gene products in a correspondingnormal sample, the presence of aberrant 24P4C12 gene products in thetest sample relative to the normal sample providing an indication ofdisregulated cell growth within the individual.
 32. A method ofdiagnosing the presence of cancer in an individual comprising: (a)determining the level of 24P4C12 mRNA expressed in a test sampleobtained from the individual; and (b) comparing the level so determinedto the level of 24P4C12 mRNA expressed in a comparable known normaltissue sample, the presence of elevated 24P4C12 mRNA expression in thetest sample relative to the normal tissue sample providing an indicationof the presence of cancer.
 33. A method of diagnosing the presence ofcancer in an individual comprising: (a) determining the level of 24P4C12protein expressed in a test sample obtained from the individual; and (b)comparing the level so determined to the level of 24P4C12 proteinexpressed in a comparable known normal tissue sample, the presence ofelevated 24P4C12 protein in the test sample relative to the normaltissue sample providing an indication of the presence of cancer.
 34. Themethod of claim 32 or 33, wherein the cancer is prostate cancer, and thetest and normal tissue samples are selected from the group consisting ofprostate tissue, bone tissue, lymphatic tissue, serum, blood or semen.35. A method for identifying a 24P4C12 specific binding agentcomprising: (a) contacting a candidate agent that binds 24P4C12 withH38087; and (b) determining whether the candidate agent binds H38087, alack of binding of the candidate agent to H38087 being indicative of24P4C12 specificity.
 36. A 24P4C12-specific binding agent identified bythe method of claim
 35. 37. A pharmaceutical composition comprising a24P4C12 polypeptide of claim of claim 10 or an immunogenic portionthereof, the vector of claim 26, an antisense polynucleotidecomplementary to a polynucleotide of claim 1, a ribozyme capable ofcleaving a polynucleotide of claim 1, an antibody or fragment thereof ofclaim 13, or a 24P4C12 specific binding agent of claim 36, and,optionally, a physiologically acceptable carrier.
 38. A method oftreating a patient with a cancer that expresses 24P4C12 which comprisesadministering to said patient a vector according to claim 26, such thatthe vector delivers the single chain monoclonal antibody coding sequenceto the cancer cells and the encoded single chain antibody is expressedintracellularly therein.
 39. A method of treating a patient with acancer that expresses 24P4C12 which comprises administering to saidpatient a composition of claim
 37. 40. A vaccine composition for thetreatment of a cancer expressing 24P4C12 comprising an immunogenicportion of a 24P4C12 protein and a physiologically acceptable carrier.41. A method of inhibiting the development or progression of a cancerexpressing 24P4C12 in a patient, comprising administering to the patientan effective amount of the vaccine composition of claim 40.